Expression cassettes for seed-preferential expression in plants

ABSTRACT

The present invention relates to expression cassettes comprising transcription regulating sequences with seed-preferential or seed-specific expression profiles in plants obtainable from  Arabidopsis thaliana  genes At4g12910, At1g66250, At4g00820, At2g36640, At2g34200, At3g11180, At4g00360, At2g48030, At2g38590, At1g23000, At3g15510, At3g50870, At4g00220, or At4g26320.

RELATED APPLICATIONS

This application claims benefit to European Application 04026295.8 filedNov. 5, 2004, to European Application 04029024.9 filed Dec. 8, 2004, toEuropean Application 05002262.3 filed Feb. 3, 2005, and to EuropeanApplication 05002850.5 filed Feb. 11, 2005.

FIELD OF THE INVENTION

The present invention relates to expression cassettes comprisingtranscription regulating nucleotide sequences with seed-preferential orseed-specific expression profiles in plants obtainable from Arabidopsisthaliana genes At4g12910, At1g66250, At4g00820, At2g36640, At2g34200,At3g11180, At4g00360, At2g48030, At2g38590, At1g23000, At3g15510,At3g50870, At4g00220, or At4g26320.

BACKGROUND OF THE INVENTION

Manipulation of plants to alter and/or improve phenotypiccharacteristics (such as productivity or quality) requires theexpression of heterologous genes in plant tissues. Such geneticmanipulation relies on the availability of a means to drive and tocontrol gene expression as required. For example, genetic manipulationrelies on the availability and use of suitable promoters which areeffective in plants and which regulate gene expression so as to give thedesired effect(s) in the transgenic plant.

The seed-preferential or seed-specific promoters are useful forexpressing genes as well as for producing large quantities of protein,for expressing oils or proteins of interest, e.g., antibodies, genes forincreasing the nutritional value of the seed and the like. It isadvantageous to have the choice of a variety of different promoters sothat the most suitable promoter may be selected for a particular gene,construct, cell, tissue, plant or environment. Moreover, the increasinginterest in cotransforming plants with multiple plant transcriptionunits (PTU) and the potential problems associated with using commonregulatory sequences for these purposes merit having a variety ofpromoter sequences available.

There is, therefore, a great need in the art for the identification ofnovel sequences that can be used for expression of selected transgenesin economically important plants. It is thus an objective of the presentinvention to provide new and alternative expression cassettes forseed-preferential or seed-specific expression of transgenes in plants.The objective is solved by the present invention.

SUMMARY OF THE INVENTION

Accordingly, a first embodiment of the invention relates to anexpression cassette for seed-specific or seed-preferential transcription(e.g., of an operatively linked nucleic acid sequence) in plantscomprising

-   -   i) at least one transcription regulating nucleotide sequence of        a plant gene, said plant gene selected from the group of genes        described by the GenBank Arabidopsis thaliana genome loci        At4g12910, At1g66250, At4g00820, At2g36640, At2g34200,        At3g11180, At4g00360, At2g48030, At2g38590, At1g23000,        At3g15510, At3g50870, At4g00220, or At4g26320, or a functional        equivalent thereof, and functionally linked thereto    -   ii) at least one nucleic acid sequence which is heterologous in        relation to said transcription regulating nucleotide sequence.

Preferably, the transcription regulating nucleotide sequence (or thefunctional equivalent thereof) is selected from the group of sequencesconsisting of

-   -   i) the sequences described by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27,        28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,        50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67,        68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148,        149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163,        164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180,        181, 182, 183, 184, and 185,    -   ii) a fragment of at least 50 consecutive bases of a sequence        under i) which has substantially the same promoter activity as        the corresponding transcription regulating nucleotide sequence        described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36,        37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54,        55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74,        75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152,        153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 168, 169,        170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183, 184,        or 185;    -   iii) a nucleotide sequence having substantial similarity (e.g.,        with a sequence identity of at least 40, 50, 60, to 70%, e.g.,        preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,        generally at least 80%, e.g., 81% to 84%, at least 85%, e.g.,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to        98% and 99%) to a transcription regulating nucleotide sequence        described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36,        37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54,        55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74,        75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152,        153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 168, 169,        170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183, 184,        or 185;    -   iv) a nucleotide sequence capable of hybridizing under        conditions equivalent to hybridization in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        2×SSC, 0.1% SDS at 50° C. (more desirably in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium        dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with        washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium        dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with        washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in 7%        sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C.        with washing in 0.1×SSC, 0.1% SDS at 65° C.) to a transcription        regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24,        25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44,        45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64,        65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86,        87, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161,        162, 163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178,        179, 180, 181, 182, 183, 184, or 185, or the complement thereof;    -   v) a nucleotide sequence capable of hybridizing under conditions        equivalent to hybridization in 7% sodium dodecyl sulfate (SDS),        0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS        at 50° C. (more desirably in 7% sodium dodecyl sulfate (SDS),        0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS        at 50° C., more desirably still in 7% sodium dodecyl sulfate        (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC,        0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate        (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,        0.1% SDS at 50° C., more preferably in 7% sodium dodecyl sulfate        (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,        0.1% SDS at 65° C.) to a nucleic acid comprising 50 to 200 or        more consecutive nucleotides of a transcription regulating        nucleotide sequence described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27,        28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,        50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67,        68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148,        149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163,        164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180,        181, 182, 183, 184, and 185, or the complement thereof;    -   vi) a nucleotide sequence which is the complement or reverse        complement of any of the previously mentioned nucleotide        sequences under i) to v).

The functional equivalent of the transcription regulating nucleotidesequence is obtained or obtainable from plant genomic DNA from a geneencoding a polypeptide which has at least 70% amino acid sequenceidentity to a polypeptide selected from the group described by SEQ IDNO: 14, 22, 30, 35, 49, 59, 73, 79, 83, 89, 157, 167, 177, and 187,respectively.

The expression cassette may be employed for numerous expression purposessuch as for example expression of a protein, or expression of aantisense RNA, sense or double-stranded RNA. Preferably, expression ofthe nucleic acid sequence confers to the plant an agronomically valuabletrait.

Other embodiments of the invention relate to vectors comprising anexpression cassette of the invention, and transgenic host cell ornon-human organism comprising an expression cassette or a vector of theinvention. Preferably the organism is a plant.

Another embodiment of the invention relates to a method for identifyingand/or isolating a sequence with seed-specific or seed-preferentialtranscription regulating activity characterized that said identificationand/or isolation utilizes a nucleic acid sequence encoding a amino acidsequence as described by SEQ ID NO: 14, 22, 30, 35, 49, 59, 73, 79, 83,89, 157, 167, 177, or 187 or a part of at least 15 bases thereof.Preferably the nucleic acid sequences is described by SEQ ID NO: 13, 21,29, 34, 48, 58, 72, 78, 82, 88, 156, 166, 176, or 186 or a part of atleast 15 bases thereof. More preferably, identification and/or isolationis realized by a method selected from polymerase chain reaction,hybridization, and database screening.

Another embodiment of the invention relates to a method for providing atransgenic expression cassette for seed-specific or seed-preferentialexpression comprising the steps of:

-   -   I. isolating of a seed-preferential or seed-specific        transcription regulating nucleotide sequence utilizing at least        one nucleic acid sequence or a part thereof, wherein said        sequence is encoding a polypeptide described by SEQ ID NO: 14,        22, 30, 35, 49, 59, 73, 79, 83, 89, 157, 167, 177, or 187, or a        part of at least 15 bases thereof, and    -   II. functionally linking said seed-preferential or seed-specific        transcription regulating nucleotide sequence to another        nucleotide sequence of interest, which is heterologous in        relation to said seed-preferential or seed-specific        transcription regulating nucleotide sequence.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, plant species or genera,constructs, and reagents described as such. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “avector” is a reference to one or more vectors and includes equivalentsthereof known to those skilled in the art, and so forth.

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20 per-cent up or down (higher or lower).As used herein, the word “or” means any one member of a particular listand also includes any combination of members of that list.

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNAor functional RNA, or encodes a specific protein, and which includesregulatory sequences. Genes also include non-expressed DNA segmentsthat, for example, form recognition sequences for other proteins. Genescan be obtained from a variety of sources, including cloning from asource of interest or synthesizing from known or predicted sequenceinformation, and may include sequences designed to have desiredparameters.

The term “native” or “wild type” gene refers to a gene that is presentin the genome of an untransformed cell, i.e., a cell not having a knownmutation.

A “marker gene” encodes a selectable or screenable trait.

The term “chimeric gene” refers to any gene that contains

-   -   1) DNA sequences, including regulatory and coding sequences,        that are not found together in nature, or    -   2) sequences encoding parts of proteins not naturally adjoined,        or    -   3) parts of promoters that are not naturally adjoined.

Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or compriseregulatory sequences and coding sequences derived from the same source,but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular plant to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism but that is introduced by genetransfer.

An “oligonucleotide” corresponding to a nucleotide sequence of theinvention, e.g., for use in probing or amplification reactions, may beabout 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or24, or any number between 9 and 30). Generally specific primers areupwards of 14 nucleotides in length. For optimum specificity and costeffectiveness, primers of 16 to 24 nucleotides in length may bepreferred. Those skilled in the art are well versed in the design ofprimers for use processes such as PCR. If required, probing can be donewith entire restriction fragments of the gene disclosed herein which maybe 100's or even 1000's of nucleotides in length.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein. The nucleotide sequences of the invention can beintroduced into any plant. The genes to be introduced can beconveniently used in expression cassettes for introduction andexpression in any plant of interest. Such expression cassettes willcomprise the transcriptional initiation region of the invention linkedto a nucleotide sequence of interest. Preferred promoters includeconstitutive, tissue-specific, developmental-specific, inducible and/orviral promoters, most preferred are the seed-specific orseed-preferential promoters of the invention. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the gene of interest to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselectable marker genes. The cassette will include in the 5′-3′direction of transcription, a transcriptional and translationalinitiation region, a DNA sequence of interest, and a transcriptional andtranslational termination region functional in plants. The terminationregion may be native with the transcriptional initiation region, may benative with the DNA sequence of interest, or may be derived from anothersource. Convenient termination regions are available from the Ti-plasmidof A. tumefaciens, such, as the octopine synthase and nopaline synthasetermination regions (see also, Guerineau 1991; Proudfoot 1991; Sanfacon1991; Mogen 1990; Munroe 1990; Ballas 1989; Joshi 1987).

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

A “functional RNA” refers to an antisense RNA, ribozyme, or other RNAthat is not translated.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Transcription regulating nucleotide sequence”, “regulatory sequences”,and “suitable regulatory sequences”, each refer to nucleotide sequencesinfluencing the transcription, RNA processing or stability, ortranslation of the associated (or functionally linked) nucleotidesequence to be transcribed. The transcription regulating nucleotidesequence may have various localizations with the respect to thenucleotide sequences to be transcribed. The transcription regulatingnucleotide sequence may be located upstream (5′ non-coding sequences),within, or downstream (3′ non-coding sequences) of the sequence to betranscribed (e.g., a coding sequence). The transcription regulatingnucleotide sequences may be selected from the group comprisingenhancers, promoters, translation leader sequences, introns,5′-untranslated sequences, 3′-untranslated sequences, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences, which may be a combination of syntheticand natural sequences. As is noted above, the term “transcriptionregulating nucleotide sequence” is not limited to promoters. However,preferably a transcription regulating nucleotide sequence of theinvention comprises at least one promoter sequence (e.g., a sequencelocalized upstream of the transcription start of a gene capable toinduce transcription of the downstream sequences). In one preferredembodiment the transcription regulating nucleotide sequence of theinvention comprises the promoter sequence of the corresponding geneand—optionally and preferably—the native 5′-untranslated region of saidgene. Furthermore, the 3′-untranslated region and/or the polyadenylationregion of said gene may also be employed.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner 1995).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., 1989.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

“Signal peptide” refers to the amino terminal extension of apolypeptide, which is translated in conjunction with the polypeptideforming a precursor peptide and which is required for its entrance intothe secretory pathway. The term “signal sequence” refers to a nucleotidesequence that encodes the signal peptide. The term “transit peptide” asused herein refers part of a expressed polypeptide (preferably to theamino terminal extension of a polypeptide), which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into a cell organelle (such as the plastids(e.g., chloroplasts) or mitochondria). The term “transit sequence”refers to a nucleotide sequence that encodes the transit peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements, derived from different promoters foundin nature, or even be comprised of synthetic DNA segments. A promotermay also contain DNA sequences that are involved in the binding ofprotein factors which control the effectiveness of transcriptioninitiation in response to physiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Constitutive promoter” refers to a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of theplant. Each of the transcription-activating elements do not exhibit anabsolute tissue-specificity, but mediate transcriptional activation inmost plant parts at a level of at least 1% of the level reached in thepart of the plant in which transcription is most active.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. New promoters of various types useful in plantcells are constantly being discovered, numerous examples may be found inthe compilation by Okamuro et al. (1989). Typical regulated promotersuseful in plants include but are not limited to safener-induciblepromoters, promoters derived from the tetracycline-inducible system,promoters derived from salicylate-inducible systems, promoters derivedfrom alcohol-inducible systems, promoters derived fromglucocorticoid-inducible system, promoters derived frompathogen-inducible systems, and promoters derived fromecdysone-inducible systems.

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all plant cells but only in one or more cell types inspecific organs (such as leaves or seeds), specific tissues (such asembryo or cotyledon), or specific cell types (such as leaf parenchyma orseed storage cells). These also include promoters that are temporallyregulated, such as in early or late embryogenesis, during fruit ripeningin developing seeds or fruit, in fully differentiated leaf, or at theonset of senescence.

“Inducible promoter” refers to those regulated promoters that can beturned on in one or more cell types by an external stimulus, such as achemical, light, hormone, stress, or a pathogen.

“Operably-linked” or “functionally linked” refers preferably to theassociation of nucleic acid sequences on single nucleic acid fragment sothat the function of one is affected by the other. For example, aregulatory DNA sequence is said to be “operably linked to” or“associated with” a DNA sequence that codes for an RNA or a polypeptideif the two sequences are situated such that the regulatory DNA sequenceaffects expression of the coding DNA sequence (i.e., that the codingsequence or functional RNA is under the transcriptional control of thepromoter). Coding sequences can be operably-linked to regulatorysequences in sense or antisense orientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, ORF or portion thereof, or a transgene in plants. Forexample, in the case of antisense constructs, expression may refer tothe transcription of the antisense DNA only. In addition, expressionrefers to the transcription and stable accumulation of sense (mRNA) orfunctional RNA. Expression may also refer to the production of protein.

“Specific expression” is the expression of gene products which islimited to one or a few plant tissues (spatial limitation) and/or to oneor a few plant developmental stages (temporal limitation). It isacknowledged that hardly a true specificity exists: promoters seem to bepreferably switch on in some tissues, while in other tissues there canbe no or only little activity. This phenomenon is known as leakyexpression. However, with specific expression in this invention is meantpreferable expression in one or a few plant tissues.

The “expression pattern” of a promoter (with or without enhancer) is thepattern of expression levels which shows where in the plant and in whatdevelopmental stage transcription is initiated by said promoter.Expression patterns of a set of promoters are said to be complementarywhen the expression pattern of one promoter shows little overlap withthe expression pattern of the other promoter. The level of expression ofa promoter can be determined by measuring the ‘steady state’concentration of a standard transcribed reporter mRNA. This measurementis indirect since the concentration of the reporter mRNA is dependentnot only on its synthesis rate, but also on the rate with which the mRNAis degraded. Therefore, the steady state level is the product ofsynthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixedrate when the transcribed sequences are identical, and thus this valuecan serve as a measure of synthesis rates. When promoters are comparedin this way techniques available to those skilled in the art arehybridization S1-RNAse analysis, northern blots and competitive RT-PCR.This list of techniques in no way represents all available techniques,but rather describes commonly used procedures used to analyzetranscription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promotershas revealed that there is usually no single base at which transcriptionstarts, but rather a more or less clustered set of initiation sites,each of which accounts for some start points of the mRNA. Since thisdistribution varies from promoter to promoter the sequences of thereporter mRNA in each of the populations would differ from each other.Since each mRNA species is more or less prone to degradation, no singledegradation rate can be expected for different reporter mRNAs. It hasbeen shown for various eukaryotic promoter sequences that the sequencesurrounding the initiation site (‘initiator’) plays an important role indetermining the level of RNA expression directed by that specificpromoter. This includes also part of the transcribed sequences. Thedirect fusion of promoter to reporter sequences would therefore lead tosuboptimal levels of transcription.

A commonly used procedure to analyze expression patterns and levels isthrough determination of the ‘steady state’ level of proteinaccumulation in a cell. Commonly used candidates for the reporter gene,known to those skilled in the art are beta-glucuronidase (GUS),chloramphenicol acetyl transferase (CAT) and proteins with fluorescentproperties, such as green fluorescent protein (GFP) from Aequoravictoria. In principle, however, many more proteins are suitable forthis purpose, provided the protein does not interfere with essentialplant functions. For quantification and determination of localization anumber of tools are suited. Detection systems can readily be created orare available which are based on, e.g., immunochemical, enzymatic,fluorescent detection and quantification. Protein levels can bedetermined in plant tissue extracts or in intact tissue using in situanalysis of protein expression.

Generally, individual transformed lines with one chimeric promoterreporter construct will vary in their levels of expression of thereporter gene. Also frequently observed is the phenomenon that suchtransformants do not express any detectable product (RNA or protein).The variability in expression is commonly ascribed to ‘positioneffects’, although the molecular mechanisms underlying this inactivityare usually not clear.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed (non-transgenic) cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Gene silencing” refers to homology-dependent suppression of viralgenes, transgenes, or endogenous nuclear genes. Gene silencing may betranscriptional, when the suppression is due to decreased transcriptionof the affected genes, or post-transcriptional, when the suppression isdue to increased turnover (degradation) of RNA species homologous to theaffected genes (English 1996). Gene silencing includes virus-inducedgene silencing (Ruiz et al. 1998).

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

“Homologous to” in the context of nucleotide sequence identity refers tothe similarity between the nucleotide sequence of two nucleic acidmolecules or between the amino acid sequences of two protein molecules.Estimates of such homology are provided by either DNA-DNA or DNA-RNAhybridization under conditions of stringency as is well understood bythose skilled in the art (as described in Haines and Higgins (eds.),Nucleic Acid Hybridization, IRL Press, Oxford, U.K.), or by thecomparison of sequence similarity between two nucleic acids or proteins.

The term “substantially similar” refers to nucleotide and amino acidsequences that represent functional and/or structural equivalents ofArabidopsis sequences disclosed herein.

In its broadest sense, the term “substantially similar” when used hereinwith respect to a nucleotide sequence means that the nucleotide sequenceis part of a gene which encodes a polypeptide having substantially thesame structure and function as a polypeptide encoded by a gene for thereference nucleotide sequence, e.g., the nucleotide sequence comprises apromoter from a gene that is the ortholog of the gene corresponding tothe reference nucleotide sequence, as well as promoter sequences thatare structurally related the promoter sequences particularly exemplifiedherein, i.e., the substantially similar promoter sequences hybridize tothe complement of the promoter sequences exemplified herein under highor very high stringency conditions. For example, altered nucleotidesequences which simply reflect the degeneracy of the genetic code butnonetheless encode amino acid sequences that are identical to aparticular amino acid sequence are substantially similar to theparticular sequences. The term “substantially similar” also includesnucleotide sequences wherein the sequence has been modified, forexample, to optimize expression in particular cells, as well asnucleotide sequences encoding a variant polypeptide having one or moreamino acid substitutions relative to the (unmodified) polypeptideencoded by the reference sequence, which substitution(s) does not alterthe activity of the variant polypeptide relative to the unmodifiedpolypeptide.

In its broadest sense, the term “substantially similar” when used hereinwith respect to polypeptide means that the polypeptide has substantiallythe same structure and function as the reference polypeptide. Inaddition, amino acid sequences that are substantially similar to aparticular sequence are those wherein overall amino acid identity is atleast 65% or greater to the instant sequences. Modifications that resultin equivalent nucleotide or amino acid sequences are well within theroutine skill in the art. The percentage of amino acid sequence identitybetween the substantially similar and the reference polypeptide is atleast 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, andeven 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to atleast 99%, wherein the reference polypeptide is an Arabidopsispolypeptide encoded by a gene with a promoter having any one of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23,24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150,151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 168,169, 170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183, 184, or185, a nucleotide sequence comprising an open reading frame having anyone of SEQ ID NOs: 13, 21, 29, 34, 48, 58, 72, 78, 82, 88, 156, 166,176, or 186, which encodes one of SEQ ID NOs: 14, 22, 30, 35, 49, 59,73, 79, 83, 89, 157, 167, 177, or 187. One indication that twopolypeptides are substantially similar to each other, besides havingsubstantially the same function, is that an agent, e.g., an antibody,which specifically binds to one of the polypeptides, also specificallybinds to the other.

Sequence comparisons maybe carried out using a Smith-Waterman sequencealignment algorithm (see e.g., Waterman (1995)). The localS program,version 1.16, is preferably used with following parameters: match: 1,mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.

Moreover, a nucleotide sequence that is “substantially similar” to areference nucleotide sequence is said to be “equivalent” to thereference nucleotide sequence. The skilled artisan recognizes thatequivalent nucleotide sequences encompassed by this invention can alsobe defined by their ability to hybridize, under low, moderate and/orstringent conditions (e.g., 0.1×SSC, 0.1% SDS, 65° C.), with thenucleotide sequences that are within the literal scope of the instantclaims.

What is meant by “substantially the same activity” when used inreference to a polynucleotide or polypeptide fragment is that thefragment has at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, up to at least 99% of the activity of the full lengthpolynucleotide or full length polypeptide.

“Target gene” refers to a gene on the replicon that expresses thedesired target coding sequence, functional RNA, or protein. The targetgene is not essential for replicon replication. Additionally, targetgenes may comprise native non-viral genes inserted into a non-nativeorganism, or chimeric genes, and will be under the control of suitableregulatory sequences. Thus, the regulatory sequences in the target genemay come from any source, including the virus. Target genes may includecoding sequences that are either heterologous or homologous to the genesof a particular plant to be transformed. However, target genes do notinclude native viral genes. Typical target genes include, but are notlimited to genes encoding a structural protein, a seed storage protein,a protein that conveys herbicide resistance, and a protein that conveysinsect resistance. Proteins encoded by target genes are known as“foreign proteins”. The expression of a target gene in a plant willtypically produce an altered plant trait.

The term “altered plant trait” means any phenotypic or genotypic changein a transgenic plant relative to the wild-type or non-transgenic planthost.

“Replication gene” refers to a gene encoding a viral replicationprotein. In addition to the ORF of the replication protein, thereplication gene may also contain other overlapping or non-overlappingORF(s), as are found in viral sequences in nature. While not essentialfor replication, these additional ORFs may enhance replication and/orviral DNA accumulation. Examples of such additional ORFs are AC3 and AL3in ACMV and TGMV geminiviruses, respectively.

“Chimeric trans-acting replication gene” refers either to a replicationgene in which the coding sequence of a replication protein is under thecontrol of a regulated plant promoter other than that in the nativeviral replication gene, or a modified native viral replication gene, forexample, in which a site specific sequence(s) is inserted in the 5′transcribed but untranslated region. Such chimeric genes also includeinsertion of the known sites of replication protein binding between thepromoter and the transcription start site that attenuate transcriptionof viral replication protein gene.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.Examples of methods of transformation of plants and plant cells includeAgrobacterium-mediated transformation (De Blaere 1987) and particlebombardment technology (U.S. Pat. No. 4,945,050). Whole plants may beregenerated from transgenic cells by methods well known to the skilledartisan (see, for example, Fromm 1990).

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome generally known in the art and are disclosed(Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999. Knownmethods of PCR include, but are not limited to, methods using pairedprimers, nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially mismatchedprimers, and the like. For example, “transformed,” “transformant,” and“transgenic” plants or calli have been through the transformationprocess and contain a foreign gene integrated into their chromosome. Theterm “untransformed” refers to normal plants that have not been throughthe transformation process.

“Transiently transformed” refers to cells in which transgenes andforeign DNA have been introduced (for example, by such methods asAgrobacterium-mediated transformation or biolistic bombardment), but notselected for stable maintenance.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“Transient expression” refers to expression in cells in which a virus ora transgene is introduced by viral infection or by such methods asAgrobacterium-mediated transformation, electroporation, or biolisticbombardment, but not selected for its stable maintenance.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Primary transformant” and “T0 generation” refer to transgenic plantsthat are of the same genetic generation as the tissue which wasinitially transformed (i.e., not having gone through meiosis andfertilization since transformation).

“Secondary transformants” and the “T1, T2, T3, etc. generations” referto transgenic plants derived from primary transformants through one ormore meiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

“Genome” refers to the complete genetic material of an organism.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer 1991; Ohtsuka 1985; Rossolini 1994). A“nucleic acid fragment” is a fraction of a given nucleic acid molecule.In higher plants, deoxyribonucleic acid (DNA) is the genetic materialwhile ribonucleic acid (RNA) is involved in the transfer of informationcontained within DNA into proteins. The term “nucleotide sequence”refers to a polymer of DNA or RNA which can be single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases capable of incorporation into DNA or RNA polymers. Theterms “nucleic acid” or “nucleic acid sequence” may also be usedinterchangeably with gene, cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” DNA molecule or an “isolated” or “purified”polypeptide is a DNA molecule or polypeptide that, by the hand of man,exists apart from its native environment and is therefore not a productof nature. An isolated DNA molecule or polypeptide may exist in apurified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. Preferably, an “isolated” nucleic acid is free of sequences(preferably protein encoding sequences) that naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. A protein that is substantially free of cellular materialincludes preparations of protein or polypeptide having less than about30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When theprotein of the invention, or biologically active portion thereof, isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-protein of interest chemicals.

The nucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant (variant) forms. Such variantswill continue to possess the desired activity, i.e., either promoteractivity or the activity of the product encoded by the open readingframe of the non-variant nucleotide sequence.

The term “variant” with respect to a sequence (e.g., a polypeptide ornucleic acid sequence such as—for example—a transcription regulatingnucleotide sequence of the invention) is intended to mean substantiallysimilar sequences. For nucleotide sequences comprising an open readingframe, variants include those sequences that, because of the degeneracyof the genetic code, encode the identical amino acid sequence of thenative protein. Naturally occurring allelic variants such as these canbe identified with the use of well-known molecular biology techniques,as, for example, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis and for open reading frames, encode thenative protein, as well as those that encode a polypeptide having aminoacid substitutions relative to the native protein. Generally, nucleotidesequence variants of the invention will have at least 40, 50, 60, to70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%nucleotide sequence identity to the native (wild type or endogenous)nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

The nucleic acid molecules of the invention can be “optimized” forenhanced expression in plants of interest (see, for example, WO91/16432; Perlak 1991; Murray 1989). In this manner, the open readingframes in genes or gene fragments can be synthesized utilizingplant-preferred codons (see, for example, Campbell & Gowri, 1990 for adiscussion of host-preferred codon usage). Thus, the nucleotidesequences can be optimized for expression in any plant. It is recognizedthat all or any part of the gene sequence may be optimized or synthetic.That is, synthetic or partially optimized sequences may also be used.Variant nucleotide sequences and proteins also encompass, sequences andprotein derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different codingsequences can be manipulated to create a new polypeptide possessing thedesired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. Strategies for such DNA shuffling are known in the art (see, forexample, Stemmer 1994; Stemmer 1994; Crameri 1997; Moore 1997; Zhang1997; Crameri 1998; and U.S. Pat. Nos. 5,605,793 and 5,837,458).

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

Thus, the polypeptides may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions. Methods forsuch manipulations are generally known in the art. For example, aminoacid sequence variants of the polypeptides can be prepared by mutationsin the DNA. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art (see, for example, Kunkel 1985; Kunkel 1987;U.S. Pat. No. 4,873,192; Walker & Gaastra, 1983 and the references citedtherein). Guidance as to appropriate amino acid substitutions that donot affect biological activity of the protein of interest may be foundin the model of Dayhoff et al. (1978). Conservative substitutions, suchas exchanging one amino acid with another having similar properties, arepreferred. Individual substitutions deletions or additions that alter,add or delete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). See also, Creighton, 1984. Inaddition, individual substitutions, deletions or additions which alter,add or delete a single amino acid or a small percentage of amino acidsin an encoded sequence are also “conservatively modified variations.”

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to anucleotide sequence of interest, which is—optionally—operably linked totermination signals and/or other regulatory elements. An expressioncassette may also comprise sequences required for proper translation ofthe nucleotide sequence. The coding region usually codes for a proteinof interest but may also code for a functional RNA of interest, forexample antisense RNA or a nontranslated RNA, in the sense or antisensedirection. The expression cassette comprising the nucleotide sequence ofinterest may be chimeric, meaning that at least one of its components isheterologous with respect to at least one of its other components. Theexpression cassette may also be one, which is naturally occurring buthas been obtained in a recombinant form useful for heterologousexpression. An expression cassette may be assembled entirelyextracellularly (e.g., by recombinant cloning techniques). However, anexpression cassette may also be assembled using in part endogenouscomponents. For example, an expression cassette may be obtained byplacing (or inserting) a promoter sequence upstream of an endogenoussequence, which thereby becomes functionally linked and controlled bysaid promoter sequences. Likewise, a nucleic acid sequence to beexpressed may be placed (or inserted) downstream of an endogenouspromoter sequence thereby forming an expression cassette. The expressionof the nucleotide sequence in the expression cassette may be under thecontrol of a constitutive promoter or of an inducible promoter whichinitiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,the promoter can also be specific to a particular tissue or organ orstage of development (e.g., the seed-specific or seed-preferentialpromoters of the invention).

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

A “transgenic plant” is a plant having one or more plant cells thatcontain an expression vector.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

-   -   (a) As used herein, “reference sequence” is a defined sequence        used as a basis for sequence comparison. A reference sequence        may be a subset or the entirety of a specified sequence; for        example, as a segment of a full length cDNA or gene sequence, or        the complete cDNA or gene sequence.    -   (b) As used herein, “comparison window” makes reference to a        contiguous and specified segment of a polynucleotide sequence,        wherein the polynucleotide sequence in the comparison window may        comprise additions or deletions (i.e., gaps) compared to the        reference sequence (which does not comprise additions or        deletions) for optimal alignment of the two sequences.        Generally, the comparison window is at least 20 contiguous        nucleotides in length, and optionally can be 30, 40, 50, 100, or        longer. Those of skill in the art understand that to avoid a        high similarity to a reference sequence due to inclusion of gaps        in the polynucleotide sequence a gap penalty is typically        introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, 1988; the local homology algorithm of Smith et al.1981; the homology alignment algorithm of Needleman and Wunsch 1970; thesearch-for-similarity-method of Pearson and Lipman 1988; the algorithmof Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described (Higgins 1988, 1989;Corpet 1988; Huang 1992; Pearson 1994). The ALIGN program is based onthe algorithm of Myers and Miller, supra. The BLAST programs of Altschulet al., 1990, are based on the algorithm of Karlin and Altschul, supra.

Software for performing BLAST analyses is publicly available though theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul 1990). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative aligmnentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. 1997.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et at, supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.BLASTN for nucleotide sequences, BLASTX for proteins) can be used. TheBLASTN program (for nucleotide sequences) uses as defaults a wordlength(W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). Alignmentmay also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

-   -   (c) As used herein, “sequence identity” or “identity” in the        context of two nucleic acid or polypeptide sequences makes        reference to the residues in the two sequences that are the same        when aligned for maximum correspondence over a specified        comparison window. When percentage of sequence identity is used        in reference to proteins it is recognized that residue positions        which are not identical often differ by conservative amino acid        substitutions, where amino acid residues are substituted for        other amino acid residues with similar chemical properties        (e.g., charge or hydrophobicity) and therefore do not change the        functional properties of the molecule. When sequences differ in        conservative substitutions, the percent sequence identity may be        adjusted upwards to correct for the conservative nature of the        substitution. Sequences that differ by such conservative        substitutions are said to have “sequence similarity” or        “similarity.” Means for making this adjustment are well known to        those of skill in the art. Typically this involves scoring a        conservative substitution as a partial rather than a full        mismatch, thereby increasing the percentage sequence identity.        Thus, for example, where an identical amino acid is given a        score of 1 and a non-conservative substitution is given a score        of zero, a conservative substitution is given a score between        zero and 1. The scoring of conservative substitutions is        calculated, e.g., as implemented in the program PC/GENE        (Intelligenetics, Mountain View, Calif.).    -   (d) As used herein, “percentage of sequence identity” means the        value determined by comparing two optimally aligned sequences        over a comparison window, wherein the portion of the        polynucleotide sequence in the comparison window may comprise        additions or deletions (i.e., gaps) as compared to the reference        sequence (which does not comprise additions or deletions) for        optimal alignment of the two sequences. The percentage is        calculated by determining the number of positions at which the        identical nucleic acid base or amino acid residue occurs in both        sequences to yield the number of matched positions, dividing the        number of matched positions by the total number of positions in        the window of comparison, and multiplying the result by 100 to        yield the percentage of sequence identity.    -   (e) (i) The term “substantial identity” or “substantial        similarity” of polynucleotide sequences for a protein encoding        sequence means that a polynucleotide comprises a sequence that        has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or        79%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,        88%, or 89%, more preferably at least 90%, 91%, 92%, 93%, or        94%, and most preferably at least 95%, 96%, 97%, 98%, or 99%        sequence identity, compared to a reference sequence using one of        the alignment programs described using standard parameters. The        term “substantial identity” or “substantial similarity” of        polynucleotide sequences for promoter sequence means (as        described above for variants) that a polynucleotide comprises a        sequence that has at least 40, 50, 60, to 70%, e.g., preferably        71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at        least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%,        89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%        nucleotide sequence identity compared to a reference sequence        using one of the alignment programs described using standard        parameters. One of skill in the art will recognize that these        values can be appropriately adjusted to determine corresponding        identity of proteins encoded by two nucleotide sequences by        taking into account codon degeneracy, amino acid similarity,        reading frame positioning, and the like. Substantial identity of        amino acid sequences for these purposes normally means sequence        identity of at least 70%, more preferably at least 80%, 90%, and        most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

-   -   -   (ii) The term “substantial identity” in the context of a            peptide indicates that a peptide comprises a sequence with            at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or            79%, preferably 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,            or 89%, more preferably at least 90%, 91%, 92%, 93%, or 94%,            or even more preferably, 95%, 96%, 97%, 98% or 99%, sequence            identity to the reference sequence over a specified            comparison window. Preferably, optimal alignment is            conducted using the homology alignment algorithm of            Needleman and Wunsch (1970). An indication that two peptide            sequences are substantially identical is that one peptide is            immunologically reactive with antibodies raised against the            second peptide. Thus, a peptide is substantially identical            to a second peptide, for example, where the two peptides            differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridization are sequence dependent, andare different under different environmental parameters. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, 1984:T _(m)=81.5° C.+16.6(log₁₀ M)+0.41(% GC)−0.61(% form)−500/Lwhere M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the thermal melting point I for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point I; moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the thermal melting point I; low stringency conditions canutilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point I. Using the equation,hybridization and wash compositions, and desired T, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a T of less than 45° C. (aqueous solution) or 32°C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, 1993.Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point T_(m)for the specific sequence at a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4 to 6×SSC at 40° C. for15 minutes. For short probes (e.g., about 10 to 50 nucleotides),stringent conditions typically involve salt concentrations of less thanabout 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration(or other salts) at pH 7.0 to 8.3, and the temperature is typically atleast about 30° C. and at least about 60° C. for long robes (e.g., >50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Nucleic acids that do not hybridize to eachother under stringent conditions are still substantially identical ifthe proteins that they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone orthologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

“DNA shuffling” is a method to introduce mutations or rearrangements,preferably randomly, in a DNA molecule or to generate exchanges of DNAsequences between two or more DNA molecules, preferably randomly. TheDNA molecule resulting from DNA shuffling is a shuffled DNA moleculethat is a non-naturally occurring DNA molecule derived from at least onetemplate DNA molecule. The shuffled DNA preferably encodes a variantpolypeptide modified with respect to the polypeptide encoded by thetemplate DNA, and may have an altered biological activity with respectto the polypeptide encoded by the template DNA.

“Recombinant DNA molecule’ is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook etal., 1989.

The word “plant” refers to any plant, particularly to agronomicallyuseful plants (e.g., seed plants), and “plant cell” is a structural andphysiological unit of the plant, which comprises a cell wall but mayalso refer to a protoplast. The plant cell may be in form of an isolatedsingle cell or a cultured cell, or as a part of higher organized unitsuch as, for example, a plant tissue, or a plant organ differentiatedinto a structure that is present at any stage of a plant's development.Such structures include one or more plant organs including, but are notlimited to, fruit, shoot, stem, leaf, flower petal, etc. Preferably, theterm “plant” includes whole plants, shoot vegetative organs/structures(e.g. leaves, stems and tubers), roots, flowers and floralorgans/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seeds (including embryo, endosperm, and seed coat)and fruits (the mature ovary), plant tissues (e.g. vascular tissue,ground tissue, and the like) and cells (e.g. guard cells, egg cells,trichomes and the like), and progeny of same.

The class of plants that can be used in the method of the invention isgenerally as broad as the class of higher and lower plants amenable totransformation techniques, including angiosperms (monocotyledonous anddicotyledonous plants), gymnosperms, ferns, and multicellular algae. Itincludes plants of a variety of ploidy levels, including aneuploid,polyploid, diploid, haploid and hemizygous. Included within the scope ofthe invention are all genera and species of higher and lower plants ofthe plant kingdom.

Included are furthermore the mature plants, seed, shoots and seedlings,and parts, propagation material (for example seeds and fruit) andcultures, for example cell cultures, derived therefrom. Preferred areplants and plant materials of the following plant families:Amaranthaceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae,Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae,Linaceae, Malvaceae, Rosaceae, Saxifragaceae, Scrophulariaceae,Solanaceae, Tetragoniaceae.

Annual, perennial, monocotyledonous and dicotyledonous plants arepreferred host organisms for the generation of transgenic plants. Theuse of the recombination system, or method according to the invention isfurthermore advantageous in all ornamental plants, forestry, fruit, orornamental trees, flowers, cut flowers, shrubs or turf. Said plant mayinclude—but shall not be limited to—bryophytes such as, for example,Hepaticae (hepaticas) and Musci (mosses); pteridophytes such as ferns,horsetail and clubmosses; gymnosperms such as conifers, cycads, ginkgoand Gnetaeae; algae such as Chlorophyceae, Phaeophpyceae, Rhodophyceae,Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) andEuglenophyceae.

Plants for the purposes of the invention may comprise the families ofthe Rosaceae such as rose, Ericaceae such as rhododendrons and azaleas,Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such aspinks, Solanaceae such as petunias, Gesneriaceae such as African violet,Balsaminaceae such as touch-me-not, Orchidaceae such as orchids,Iridaceae such as gladioli, iris, freesia and crocus, Compositae such asmarigold, Geraniaceae such as geraniums, Liliaceae such as Drachaena,Moraceae such as ficus, Araceae such as philodendron and many others.

The transgenic plants according to the invention are furthermoreselected in particular from among dicotyledonous crop plants such as,for example, from the families of the Leguminosae such as pea, alfalfaand soybean; the family of the Umbelliferae, particularly the genusDaucus (very particularly the species carota (carrot)) and Apium (veryparticularly the species graveolens var. dulce (celery)) and manyothers; the family of the Solanaceae, particularly the genusLycopersicon, very particularly the species esculentum (tomato) and thegenus Solanum, very particularly the species tuberosum (potato) andmelongena (aubergine), tobacco and many others; and the genus Capsicum,very particularly the species annum (pepper) and many others; the familyof the Leguminosae, particularly the genus Glycine, very particularlythe species max (soybean) and many others; and the family of theCruciferae, particularly the genus Brassica, very particularly thespecies napus (oilseed rape), campestris (beet), oleracea cv Tastie(cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor(broccoli); and the genus Arabidopsis, very particularly the speciesthaliana and many others; the family of the Compositae, particularly thegenus Lactuca, very particularly the species sativa (lettuce) and manyothers.

The transgenic plants according to the invention may be selected amongmonocotyledonous crop plants, such as, for example, cereals such aswheat, barley, sorghum and millet, rye, triticale, maize, rice or oats,and sugarcane. Further preferred are trees such as apple, pear, quince,plum, cherry, peach, nectarine, apricot, papaya, mango, and other woodyspecies including coniferous and deciduous trees such as poplar, pine,sequoia, cedar, oak, etc. Especially preferred are Arabidopsis thaliana,Nicotiana tabacum, oilseed rape, soybean, corn (maize), wheat, Linumusitatissimum (linseed and fax), Camelina sativa, Brassica juncea,potato and tagetes.

“Significant increase” is an increase that is larger than the margin oferror inherent in the measurement technique, preferably an increase byabout 2-fold or greater.

“Significantly less” means that the decrease is larger than the marginof error inherent in the measurement technique, preferably a decrease byabout 2-fold or greater.

DETAILED DESCRIPTION OF THE INVENTION

The present invention thus provides for isolated nucleic acid moleculescomprising a plant nucleotide sequence that directs seed-preferential orseed-specific transcription of an operably linked nucleic acid fragmentin a plant cell.

Specifically, the present invention provides transgenic expressioncassettes for regulating seed-preferential or seed-specific expression(or transcription) in plants comprising

-   -   i) at least one transcription regulating nucleotide sequence of        a plant gene, said plant gene selected from the group of genes        described by the GenBank Arabidopsis thaliana genome locii        At4g12910, At1g66250, At4g00820, At2g36640, At2g34200,        At3g11180, At4g00360, At2g48030, At2g38590, At1g23000,        At3g15510, At3g50870, At4g00220, or At4g26320, or a functional        equivalent thereof, and functionally linked thereto    -   ii) at least one nucleic acid sequence which is heterologous in        relation to said transcription regulating nucleotide sequence.

The seed-preferential or seed-specific promoters may be useful forexpressing genes as well as for producing large quantities of protein,for expressing oils or proteins of interest, e.g., antibodies, genes forincreasing the nutritional value of the seed and the like.

The term “seed” in the context of the inventions means a seed of a plantin any stage of its development i.e. starting from the fusion of pollenand oocyte, continuing over the embryo stage and the stage of thedormant seed, until the germinating seed, ending with early seedlingorgans, as e.g. cotyledons and hypocotyl.

“Seed-specific transcription” in the context of this invention means thetranscription of a nucleic acid sequence by a transcription regulatingelement in a way that transcription of said nucleic acid sequence inseeds contribute to more than 90%, preferably more than 95%, morepreferably more than 99% of the entire quantity of the RNA transcribedfrom said nucleic acid sequence in the entire plant during any of itsdevelopmental stage. The transcription regulating nucleotide sequencesdesignated pSUK19, pSUK29, pSUK298, pSUK352 and their respective shorterand longer variants are considered to be seed-specific transcriptionregulating nucleotide sequences.

“Seed-preferential transcription” in the context of this invention meansthe transcription of a nucleic acid sequence by a transcriptionregulating element in a way that transcription of said nucleic acidsequence in seeds contribute to more than 50%, preferably more than 70%,more preferably more than 80% of the entire quantity of the RNAtranscribed from said nucleic acid sequence in the entire plant duringany of its developmental stage. The transcription regulating nucleotidesequences designated (pSUK222, pSUK224, pSUK226), (pSUK322, pSUK324),(pSUK250, pSUK252), (pSUK28, pSUK30), pSUK300, (pSUK292, pSUK294,pSUK296), pSUK164, (pSUK40, pSUK42), (pSUK48, pSUK50), (pSUK96, pSUK98),(pSUK152, pSUK154) and their respective shorter and longer variants areconsidered to be seed-preferential transcription regulating nucleotidesequences (transcription regulating nucleotide sequences in brackets arevariants from one specific gene locus).

Preferably a transcription regulating nucleotide sequence of theinvention comprises at least one promoter sequence of the respectivegene (e.g., a sequence localized upstream of the transcription start ofthe respective gene capable to induce transcription of the downstreamsequences). Said transcription regulating nucleotide sequence maycomprise the promoter sequence of said genes but may further compriseother elements such as the 5′-untranslated sequence, enhancer, intronsetc. Preferably, said promoter sequence directs seed-preferential orseed-specific transcription of an operably linked nucleic acid segmentin a plant or plant cell e.g., a linked plant DNA comprising an openreading frame for a structural or regulatory gene.

The following Table 1 illustrates the genes from which the promoters ofthe invention are preferably isolated, the function of said genes, thecDNA encoded by said genes, and the protein (ORF) encoded by said genes.

TABLE 1 Genes from which the promoters of the invention are preferablyisolated, putative function of said genes, cDNA and the protein encodedby said genes. Promotor mRNA locus ID Proteine ID Gene Locus Putativefunction SEQ ID cDNA SEQ ID Protein SEQ ID At4g12910 serinecarboxypeptidase I SEQ ID NO: NM_117360 NP_193027.2 precursor-likeprotein 1, 2, 3, 4, 5, 6, 7, SEQ ID NO: 13 SEQ ID NO: 14 8, 9, 10, 11,12 At1g66250 glycosyl hydrolase family SEQ ID NO: NM_105296 NP_176799.217 protein 15, 16, 17, 18, 19, SEQ ID NO: 21 SEQ ID NO: 22 20 At4g00820calmodulin-binding protein- SEQ ID NO: NM_116308 NP_567191.2 relatedprotein 23, 24, 25, 26, 27, SEQ ID NO: 29 SEQ ID NO: 30 28 At2g36640late embryogenesis abundant SEQ ID NO: NM_129219 NP_181202.1 protein(ECP63)/ 31, 32, 33 SEQ ID NO: 34 SEQ ID NO: 35 LEA protein At2g34200Zinc finger (C3HC4-type SEQ ID NO: NM_128971 NP_180967.2 RING finger)family protein 36, 37, 38, 39, 40, SEQ ID NO: 48 SEQ ID NO: 49 41, 42,43, 44, 45, 46, 47 At3g11180 oxidoreductase, 2OG- SEQ ID NO: NM_111954NP_187728.1 Fe(II) oxygenase family 50, 51, 52, 53, 54, SEQ ID NO: 58SEQ ID NO: 59 protein 55, 56, 57 At4g00360 putative cytochrome P450 SEQID NO: NM_116260 NP_191946.1 60, 61, 62, 63, 64, SEQ ID NO: 72 SEQ IDNO: 73 65, 66, 67, 68, 69 70, 71 At2g48030 endonuclease/exonuclease/ SEQID NO: NM_130370 NP_566121.1 phosphatase family 74, 75, 76, 77 SEQ IDNO: 78 SEQ ID NO: 79 protein At2g38590 F-box family protein SEQ ID NO:NM_129416 NP_181393.1 80, 81 SEQ ID NO: 82 SEQ ID NO: 83 At1g23000heavy-metal-associated SEQ ID NO: NM_102148 NP_173713.1domain-containing protein 84, 85, 86, 87 SEQ ID NO: 88 SEQ ID NO: 89At3g15510 encoding Arabidopsis SEQ ID NO: NM_112419 NP_188170.1 thalianano apical meristem 148, 149, 150, 151, SEQ ID NO: 156 SEQ ID NO: 157(NAM) family protein 152, 153, 154, 155 (NAC2) At3g50870 encodingArabidopsis SEQ ID NO: NM_114947 NP_566939.1 thaliana zinc finger (GATA158, 159, 160, 161, SEQ ID NO: 166 SEQ ID NO: 167 type) family protein162, 163, 164, 165 At4g00220 encoding Arabidopsis SEQ ID NO: NM_116239NP_191933.1 thaliana LOB domain protein 168, 169, 170, 171, SEQ ID NO:176 SEQ ID NO: 177 30/lateral organ 172, 173, 174, 175 boundaries domainprotein 30 (LBD30) At4g26320 encoding Arabidopsis SEQ ID NO: NM_118765NP_194362.1 thaliana arabinogalactan- 178, 179, 180, 181, SEQ ID NO: 186SEQ ID NO: 187 protein (AGP13) 182, 183, 184, 185

Preferably the transcription regulating nucleotide sequence (or thefunctional equivalent thereof) is selected from the group of sequencesconsisting of

-   -   i) the sequences described by SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7,        8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27,        28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,        50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67,        68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87, 148,        149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163,        164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180,        181, 182, 183, 184, and 185,    -   ii) a fragment of at least 50 consecutive bases of a sequence        under i) which has substantially the same promoter activity as        the corresponding transcription regulating nucleotide sequence        described by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17,        18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39,        40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57,        60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77,        80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155,        158, 159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172,        173, 174, 175, 178, 179, 180, 181, 182, 183, 184, or 185;    -   iii) a nucleotide sequence having substantial similarity (e.g.,        with a sequence identity of at least 40, 50, 60, to 70%, e.g.,        preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,        generally at least 80%, e.g., 81% to 84%, at least 85%, e.g.,        86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to        98%, and 99%) to a transcription regulating nucleotide sequence        described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36,        37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54,        55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74,        75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152,        153, 154, 155, 158, 159, 160, 161, 162, 163, 164, 165, 168, 169,        170, 171, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183, 184,        or 185;    -   iv) a nucleotide sequence capable of hybridizing under        conditions equivalent to hybridization in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        2×SSC, 0.1% SDS at 50° C. (more desirably in 7% sodium dodecyl        sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in        1×SSC, 0.1% SDS at 50° C., more desirably still in 7% sodium        dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with        washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodium        dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with        washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in 7%        sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C.        with washing in 0.1×SSC, 0.1% SDS at 65° C.) to a transcription        regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24,        25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44,        45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64,        65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86,        87, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161,        162, 163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178,        179, 180, 181, 182, 183, 184, or 185, or the complement thereof;    -   v) a nucleotide sequence capable of hybridizing under conditions        equivalent to hybridization in 7% sodium dodecyl sulfate (SDS),        0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS        at 50° C. (more desirably in 7% sodium dodecyl sulfate (SDS),        0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS        at 50° C., more desirably still in 7% sodium dodecyl sulfate        (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC,        0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate        (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,        0.1% SDS at 50° C., more preferably in 7% sodium dodecyl sulfate        (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,        0.1% SDS at 65° C.) to a nucleic acid comprising 50 to 200 or        more consecutive nucleotides (preferably at least 100, 200, or        300, more preferably 400, 500, or 600, most preferably at least        700, 800, or 900 consecutive nucleotides) of a transcription        regulating nucleotide sequence described by SEQ ID NO: 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24,        25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44,        45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64,        65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86,        87, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161,        162, 163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178,        179, 180, 181, 182, 183, 184, or 185, or the complement thereof;    -   vi) a nucleotide sequence which is the complement or reverse        complement of any of the previously mentioned nucleotide        sequences under i) to v).

A functional equivalent of the transcription regulating nucleotidesequence can also be obtained or is obtainable from plant genomic DNAfrom a gene encoding a polypeptide which is substantially similar andpreferably has at least 70%, preferably 80%, more preferably 90%, mostpreferably 95% amino acid sequence identity to a polypeptide encoded byan Arabidopsis thaliana gene comprising any one of SEQ ID NOs: 14, 22,30, 35, 49, 59, 73, 79, 83, 89, 157, 167, 177, or 187, respectively, ora fragment of said transcription regulating nucleotide sequence whichexhibits promoter activity in a seed-preferential or seed-specificfashion.

The activity of a transcription regulating nucleotide sequence isconsidered equivalent if transcription is initiated in aseed-preferential or seed-specific fashion (as defined above). Suchexpression profile is preferably demonstrated using reporter genesoperably linked to said transcription regulating nucleotide sequence.Preferred reporter genes (Schenborn 1999) in this context are greenfluorescence protein (GFP) (Chui 1996; Leffel 1997), chloramphenicoltransferase, luciferase (Millar 1992), β-glucuronidase orβ-galactosidase. Especially preferred is β-glucuronidase (Jefferson1987).

Beside this the transcription regulating activity of a functionequivalent may vary from the activity of its parent sequence, especiallywith respect to expression level. The expression level may be higher orlower than the expression level of the parent sequence. Both derivationsmay be advantageous depending on the nucleic acid sequence of interestto be expressed. Preferred are such functional equivalent sequences,which—in comparison with its parent sequence—does, not derivate from theexpression level of said parent sequence by more than 50%, preferably25%, more preferably 10% (as to be preferably judged by either mRNAexpression or protein (e.g., reporter gene) expression). Furthermorepreferred are equivalent sequences which demonstrate an increasedexpression in comparison to its parent sequence, preferably an increasemy at least 50%, more preferably by at least 100%, most preferably by atleast 500%.

Preferably functional equivalent of the transcription regulatingnucleotide sequence can be obtained or is obtainable from plant genomicDNA from a gene expressing a mRNA described by a cDNA which issubstantially similar and preferably has at least 70%, preferably 80%,more preferably 90%, most preferably 95% sequence identity to a sequencedescribed by any one of SEQ ID NOs: 13, 21, 29, 34, 48, 58, 72, 78, 82,88, 156, 166, 176, or 186, respectively, or a fragment of saidtranscription regulating nucleotide sequence which exhibits promoteractivity in a seed-preferential or seed-specific fashion.

Such functional equivalent of the transcription regulating nucleotidesequence may be obtained from other plant species by using theseed-preferential or seed-specific Arabidopsis promoter sequencesdescribed herein as probes to screen for homologous structural genes inother plants by hybridization under low, moderate or stringenthybridization conditions. Regions of the seed-preferential orseed-specific promoter sequences of the present invention which areconserved among species could also be used as PCR primers to amplify asegment from a species other than Arabidopsis, and that segment used asa hybridization probe (the latter approach permitting higher stringencyscreening) or in a transcription assay to determine promoter activity.Moreover, the seed-preferential or seed-specific promoter sequencescould be employed to identify structurally related sequences in adatabase using computer algorithms.

More specifically, based on the Arabidopsis nucleic acid sequences ofthe present invention, orthologs may be identified or isolated from thegenome of any desired organism, preferably from another plant, accordingto well known techniques based on their sequence similarity to theArabidopsis nucleic acid sequences, e.g., hybridization, PCR or computergenerated sequence comparisons. For example, all or a portion of aparticular Arabidopsis nucleic acid sequence is used as a probe thatselectively hybridizes to other gene sequences present in a populationof cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen source organism. Further, suitable genomic andcDNA libraries may be prepared from any cell or tissue of an organism.Such techniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, e.g., Sambrook 1989) and amplificationby PCR using oligonucleotide primers preferably corresponding tosequence domains conserved among related polypeptide or subsequences ofthe nucleotide sequences provided herein (see, e.g., Innis 1990). Thesemethods are particularly well suited to the isolation of gene sequencesfrom organisms closely related to the organism from which the probesequence is derived. The application of these methods using theArabidopsis sequences as probes is well suited for the isolation of genesequences from any source organism, preferably other plant species. In aPCR approach, oligonucleotide primers can be designed for use in PCRreactions to amplify corresponding DNA sequences from cDNA or genomicDNA extracted from any plant of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the sequence of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989). In general, sequencesthat hybridize to the sequences disclosed herein will have at least 40%to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or moreidentity with the disclosed sequences. That is, the sequence similarityof sequences may range, sharing at least about 40% to 50%, about 60% to70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The nucleic acid molecules of the invention can also be identified by,for example, a search of known databases for genes encoding polypeptideshaving a specified amino acid sequence identity or DNA having aspecified nucleotide sequence identity. Methods of alignment ofsequences for comparison are well known in the art and are describedhereinabove.

Hence, the isolated nucleic acid molecules of the invention include theorthologs of the Arabidopsis sequences disclosed herein, i.e., thecorresponding nucleotide sequences in organisms other than Arabidopsis,including, but not limited to, plants other than Arabidopsis, preferablydicotyledonous plants, e.g., Brassica napus, alfalfa, sunflower,soybean, cotton, peanut, tobacco or sugar beet, but also cereal plantssuch as corn, wheat, rye, turfgrass, sorghum, millet, sugarcane, barleyand banana. An orthologous gene is a gene from a different species thatencodes a product having the same or similar function, e.g., catalyzingthe same reaction as a product encoded by a gene from a referenceorganism. Thus, an ortholog includes polypeptides having less than,e.g., 65% amino acid sequence identity, but which ortholog encodes apolypeptide having the same or similar function. Databases such GenBankmay be employed to identify sequences related to the Arabidopsissequences, e.g., orthologs in other dicotyledonous plants such asBrassica napus and others. Alternatively, recombinant DNA techniquessuch as hybridization or PCR may be employed to identify sequencesrelated to the Arabidopsis sequences or to clone the equivalentsequences from different Arabidopsis DNAs.

The transcription regulating nucleotide sequences of the invention ortheir functional equivalents can be obtained or isolated from any plantor non-plant source, or produced synthetically by purely chemical means.Preferred sources include, but are not limited to the plants defined inthe DEFINITION section above.

Thus, another embodiment of the invention relates to a method foridentifying and/or isolating a sequence with seed-preferential orseed-specific transcription regulating activity utilizing a nucleic acidsequence encoding a amino acid sequence as described by SEQ ID NO: 14,22, 30, 35, 49, 59, 73, 79, 83, 89, 157, 167, 177, or 187 or a partthereof. Preferred are nucleic acid sequences described by SEQ ID NO:13, 21, 29, 34, 48, 58, 72, 78, 82, 88, 156, 166, 176, or 186 or partsthereof. “Part” in this context means a nucleic acid sequence of atleast 15 bases preferably at least 25 bases, more preferably at least 50bases. The method can be based on (but is not limited to) the methodsdescribed above such as polymerase chain reaction, hybridization ordatabase screening. Preferably, this method of the invention is based ona polymerase chain reaction, wherein said nucleic acid sequence or itspart is utilized as oligonucleotide primer. The person skilled in theart is aware of several methods to amplify and isolate the promoter of agene starting from part of its coding sequence (such as, for example,part of a cDNA). Such methods may include but are not limited to methodsuch as inverse PCR (“iPCR”) or “thermal asymmetric interlaced PCR”(“TAIL PCR”).

Another embodiment of the invention is related to a method for providinga transgenic expression cassette for seed-preferential or seed-specificexpression comprising the steps of:

-   -   I. isolating of a seed-preferential or seed-specific        transcription regulating nucleotide sequence utilizing at least        one nucleic acid sequence or a part thereof, wherein said        sequence is encoding a polypeptide described by SEQ ID NO: 14,        22, 30, 35, 49, 59, 73, 79, 83, 89, 157, 167, 177, or 187, or a        part of at least 15 bases thereof, and    -   II. functionally linking said seed-preferential or seed-specific        transcription regulating nucleotide sequence to another        nucleotide sequence of interest, which is heterologous in        relation to said seed-preferential or seed-specific        transcription regulating nucleotide sequence.

Preferably, the nucleic acid sequence employed for the isolationcomprises at least 15 base, preferably at least 25 bases, morepreferably at least 50 bases of a sequence described by SEQ ID NO: 13,21, 29, 34, 48, 58, 72, 78, 82, 88, 156, 166, 176, or 186. Preferably,the isolation of the seed-preferential or seed-specific transcriptionregulating nucleotide sequence is realized by a polymerase chainreaction utilizing said nucleic acid sequence as a primer. The operablelinkage can be realized by standard cloning method known in the art suchas ligation-mediated cloning or recombination-mediated cloning.

Preferably, the transcription regulating nucleotide sequences andpromoters of the invention include a consecutive stretch of about 25 to2000, including 50 to 500 or 100 to 250, and up to 1000 or 1500,contiguous nucleotides, e.g., 40 to about 743, 60 to about 743, 125 toabout 743, 250 to about 743, 400 to about 743, 600 to about 743, of anyone of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17,18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85, 86, 87,148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162, 163,164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180, 181,182, 183, 184, and 185, or the promoter orthologs thereof, which includethe minimal promoter region.

In a particular embodiment of the invention said consecutive stretch ofabout 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743,125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743,has at least 75%, preferably 80%, more preferably 90% and mostpreferably 95%, nucleic acid sequence identity with a correspondingconsecutive stretch of about 25 to 2000, including 50 to 500 or 100 to250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about743, 600 to about 743, of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32,33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54,55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76,77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158,159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173, 174,175, 178, 179, 180, 181, 182, 183, 184, and 185, or the promoterorthologs thereof, which include the minimal promoter region. Theabove-defined stretch of contiguous nucleotides preferably comprises oneor more promoter motifs selected from the group consisting of TATA box,GC-box, CAAT-box and a transcription start site.

The transcription regulating nucleotide sequences of the invention ortheir functional equivalents are capable of driving seed-preferential orseed-specific expression of a coding sequence in a target cell,particularly in a plant cell. The promoter sequences and methodsdisclosed herein are useful in regulating seed-preferential orseed-specific expression, respectively, of any heterologous nucleotidesequence in a host plant in order to vary the phenotype of that plant.These promoters can be used with combinations of enhancer, upstreamelements, and/or activating sequences from the 5′ flanking regions ofplant expressible structural genes. Similarly the upstream element canbe used in combination with various plant promoter sequences.

The transcription regulating nucleotide sequences and promoters of theinvention are useful to modify the phenotype of a plant. Various changesin the phenotype of a transgenic plant are desirable, i.e., modifyingthe fatty acid composition in a plant, altering the amino acid contentof a plant, altering a plant's pathogen defense mechanism, and the like.These results can be achieved by providing expression of heterologousproducts or increased expression of endogenous products in plants.Alternatively, the results can be achieved by providing for a reductionof expression of one or more endogenous products, particularly enzymesor cofactors in the plant. These changes result in an alteration in thephenotype of the transformed plant.

Generally, the transcription regulating nucleotide sequences andpromoters of the invention may be employed to express a nucleic acidsegment that is operably linked to said promoter such as, for example,an open reading frame, or a portion thereof, an anti-sense sequence, asequence encoding for a double-stranded RNA sequence, or a transgene inplants.

An operable linkage may—for example—comprise an sequential arrangementof the transcription regulating nucleotide sequence of the invention(for example a sequence as described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31,32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53,54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75,76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155,158, 159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173,174, 175, 178, 179, 180, 181, 182, 183, 184, or 185) with a nucleic acidsequence to be expressed, and—optionally—additional regulatory elementssuch as for example polyadenylation or transcription terminationelements, enhancers, introns etc, in a way that the transcriptionregulating nucleotide sequence can fulfill its function in the processof expression the nucleic acid sequence of interest under theappropriate conditions. The term “appropriate conditions” meanpreferably the presence of the expression cassette in a plant cell.Preferred are arrangements, in which the nucleic acid sequence ofinterest to be expressed is placed down-stream (i.e., in 3′-direction)of the transcription regulating nucleotide sequence of the invention ina way, that both sequences are covalently linked. Optionally additionalsequences may be inserted in-between the two sequences. Such sequencesmay be for example linker or multiple cloning sites. Furthermore,sequences can be inserted coding for parts of fusion proteins (in case afusion protein of the protein encoded by the nucleic acid of interest isintended to be expressed). Preferably, the distance between the nucleicacid sequence of interest to be expressed and the transcriptionregulating nucleotide sequence of the invention is not more than 200base pairs, preferably not more than 100 base pairs, more preferably nomore than 50 base pairs.

An operable linkage in relation to any expression cassette or of theinvention may be realized by various methods known in the art,comprising both in vitro and in vivo procedure. Thus, an expressioncassette of the invention or an vector comprising such expressioncassette may by realized using standard recombination and cloningtechniques well known in the art (see e.g., Maniatis 1989; Silhavy 1984;Ausubel 1987).

An expression cassette may also be assembled by inserting atranscription regulating nucleotide sequence of the invention (forexample a sequence as described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55,56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77,80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158,159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173, 174,175, 178, 179, 180, 181, 182, 183, 184, or 185) into the plant genome.Such insertion will result in an operable linkage to a nucleic acidsequence of interest which as such already existed in the genome. By theinsertion the nucleic acid of interest is expressed in aseed-preferential or seed-specific way due to the transcriptionregulating properties of the transcription regulating nucleotidesequence. The insertion may be directed or by chance. Preferably theinsertion is directed and realized by for example homologousrecombination. By this procedure a natural promoter may be exchangedagainst the transcription regulating nucleotide sequence of theinvention, thereby modifying the expression profile of an endogenousgene. The transcription regulating nucleotide sequence may also beinserted in a way, that antisense mRNA of an endogenous gene isexpressed, thereby inducing gene silencing.

Similar, a nucleic acid sequence of interest to be expressed may byinserted into a plant genome comprising the transcription regulatingnucleotide sequence in its natural genomic environment (i.e. linked toits natural gene) in a way that the inserted sequence becomes operablylinked to the transcription regulating nucleotide sequence, therebyforming an expression cassette of the invention.

The open reading frame to be linked to the transcription regulatingnucleotide sequence of the invention may be obtained from an insectresistance gene, a disease resistance gene such as, for example, abacterial disease resistance gene, a fungal disease resistance gene, aviral disease resistance gene, a nematode disease resistance gene, aherbicide resistance gene, a gene affecting grain composition orquality, a nutrient utilization gene, a mycotoxin reduction gene, a malesterility gene, a selectable marker gene, a screenable marker gene, anegative selectable marker, a positive selectable marker, a geneaffecting plant agronomic characteristics, i.e., yield, standability,and the like, or an environment or stress resistance gene, i.e., one ormore genes that confer herbicide resistance or tolerance, insectresistance or tolerance, disease resistance or tolerance (viral,bacterial, fungal, oomycete, or nematode), stress tolerance orresistance (as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress, or oxidativestress), increased yields, food content and makeup, physical appearance,male sterility, drydown, standability, prolificacy, starch properties orquantity, oil quantity and quality, amino acid or protein composition,and the like. By “resistant” is meant a plant, which exhibitssubstantially no phenotypic changes as a consequence of agentadministration, infection with a pathogen, or exposure to stress. By“tolerant” is meant a plant, which, although it may exhibit somephenotypic changes as a consequence of infection, does not have asubstantially decreased reproductive capacity or substantially alteredmetabolism.

Seed-preferential or seed-specific transcription regulating nucleotidesequences (e.g., promoters) are useful for expressing a wide variety ofgenes including those which alter metabolic pathways, confer diseaseresistance, for protein production, e.g., antibody production, or toimprove nutrient uptake and the like. Seed-preferential or seed-specifictranscription regulating nucleotide sequences (e.g., promoters) may bemodified so as to be regulatable, e.g., inducible. The genes andtranscription regulating nucleotide sequences (e.g., promoters)described hereinabove can be used to identify orthologous genes andtheir transcription regulating nucleotide sequences (e.g., promoters)which are also likely expressed in a particular tissue and/ordevelopment manner. Moreover, the orthologous transcription regulatingnucleotide sequences (e.g., promoters) are useful to express linked openreading frames. In addition, by aligning the transcription regulatingnucleotide sequences (e.g., promoters) of these orthologs, novel ciselements can be identified that are useful to generate synthetictranscription regulating nucleotide sequences (e.g., promoters).

The expression regulating nucleotide sequences specified above may beoptionally operably linked to other suitable regulatory sequences, e.g.,a transcription terminator sequence, operator, repressor-binding site,transcription factor binding site and/or an enhancer.

The present invention further provides a recombinant vector containingthe expression cassette of the invention, and host cells comprising theexpression cassette or vector, e.g., comprising a plasmid. Theexpression cassette or vector may augment the genome of a transformedplant or may be maintained extra chromosomally. The expression cassetteor vector of the invention may be present in the nucleus, chloroplast,mitochondria and/or plastid of the cells of the plant. Preferably, theexpression cassette or vector of the invention is comprised in thechromosomal DNA of the plant nucleus. The present invention alsoprovides a transgenic plant prepared by this method, a seed from such aplant and progeny plants from such a plant including hybrids andinbreds. The expression cassette may be operatively linked to astructural gene, the open reading frame thereof, or a portion thereof.The expression cassette may further comprise a Ti plasmid and becontained in an Agrobacterium tumefaciens cell; it may be carried on amicroparticle, wherein the microparticle is suitable for ballistictransformation of a plant cell; or it may be contained in a plant cellor protoplast. Further, the expression cassette or vector can becontained in a transformed plant or cells thereof, and the plant may bea dicot or a monocot. In particular, the plant may be a dicotyledonousplant. Preferred transgenic plants are transgenic maize, soybean,barley, alfalfa, sunflower, canola, soybean, cotton, peanut, sorghum,tobacco, sugarbeet, rice, wheat, rye, turfgrass, millet, sugarcane,tomato, or potato.

The invention also provides a method of plant breeding, e.g., to preparea crossed fertile transgenic plant. The method comprises crossing afertile transgenic plant comprising a particular expression cassette ofthe invention with itself or with a second plant, e.g., one lacking theparticular expression cassette, to prepare the seed of a crossed fertiletransgenic plant comprising the particular expression cassette. The seedis then planted to obtain a crossed fertile transgenic plant. The plantmay be a monocot or a dicot. In a particular embodiment, the plant is adicotyledonous plant. The crossed fertile transgenic plant may have theparticular expression cassette inherited through a female parent orthrough a male parent. The second plant may be an inbred plant. Thecrossed fertile transgenic may be a hybrid. Also included within thepresent invention are seeds of any of these crossed fertile transgenicplants.

The transcription regulating nucleotide sequences of the inventionfurther comprise sequences which are complementary to one (hereinafter“test” sequence) which hybridizes under stringent conditions with anucleic acid molecule as described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32,33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54,55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76,77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158,159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172, 173, 174,175, 178, 179, 180, 181, 182, 183, 184, or 185 as well as RNA which istranscribed from the nucleic acid molecule. When the hybridization isperformed under stringent conditions, either the test or nucleic acidmolecule of invention is preferably supported, e.g., on a membrane orDNA chip. Thus, either a denatured test or nucleic acid molecule of theinvention is preferably first bound to a support and hybridization iseffected for a specified period of time at a temperature of, e.g.,between 55 and 70° C., in double strength citrate buffered saline (SC)containing 0.1% SDS followed by rinsing of the support at the sametemperature but with a buffer having a reduced SC concentration.Depending upon the degree of stringency required such reducedconcentration buffers are typically single strength SC containing 0.1%SDS, half strength SC containing 0.1% SDS and one-tenth strength SCcontaining 0.1% SDS. More preferably hybridization is carried out underhigh stringency conditions (as defined above).

Virtually any DNA composition may be used for delivery to recipientplant cells, e.g., dicotyledonous cells, to ultimately produce fertiletransgenic plants in accordance with the present invention. For example,DNA segments or fragments in the form of vectors and plasmids, or linearDNA segments or fragments, in some instances containing only the DNAelement to be expressed in the plant, and the like, may be employed. Theconstruction of vectors, which may be employed in conjunction with thepresent invention, will be known to those of skill of the art in lightof the present disclosure (see, e.g., Sambrook 1989; Gelvin 1990).

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes) and DNA segments for use intransforming such cells will, of course, generally comprise the cDNA,gene or genes which one desires to introduce into the cells. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even regulatory genes as desired. The DNA segment,fragment or gene chosen for cellular introduction will often encode aprotein which will be expressed in the resultant recombinant cells, suchas will result in a screenable or selectable trait and/or which willimpart an improved phenotype to the regenerated plant. However, this maynot always be the case, and the present invention also encompassestransgenic plants incorporating non-expressed transgenes.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) “shuttle” vectors,such as pW1-11 and PW1-GUS (Ugaki 1991). These vectors are capable ofautonomous replication in maize cells as well as E. coli, and as suchmay provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs 1990) that transposition of theseelements within the maize genome requires DNA replication. It is alsocontemplated that transposable elements would be useful for introducingDNA segments or fragments lacking elements necessary for selection andmaintenance of the plasmid vector in bacteria, e.g., antibioticresistance genes and origins of DNA replication. It is also proposedthat use of a transposable element such as Ac, Ds, or Mu would activelypromote integration of the desired DNA and hence increase the frequencyof stably transformed cells. The use of a transposable element such asAc, Ds, or Mu may actively promote integration of the DNA of interestand hence increase the frequency of stably transformed cells.Transposable elements may be useful to allow separation of genes ofinterest from elements necessary for selection and maintenance of aplasmid vector in bacteria or selection of a transformant. By use of atransposable element, desirable and undesirable DNA sequences may betransposed apart from each other in the genome, such that throughgenetic segregation in progeny, one may identify plants with either thedesirable undesirable DNA sequences.

The nucleotide sequence of interest linked to one or more of thetranscription regulating nucleotide sequences of the invention can, forexample, code for a ribosomal RNA, an antisense RNA or any other type ofRNA that is not translated into protein. In another preferred embodimentof the invention, said nucleotide sequence of interest is translatedinto a protein product. The transcription regulating nucleotide sequenceand/or nucleotide sequence of interest linked thereto may be ofhomologous or heterologous origin with respect to the plant to betransformed. A recombinant DNA molecule useful for introduction intoplant cells includes that which has been derived or isolated from anysource, that may be subsequently characterized as to structure, sizeand/or function, chemically altered, and later introduced into plants.An example of a nucleotide sequence or segment of interest “derived”from a source, would be a nucleotide sequence or segment that isidentified as a useful fragment within a given organism, and which isthen chemically synthesized in essentially pure form. An example of sucha nucleotide sequence or segment of interest “isolated” from a source,would be nucleotide sequence or segment that is excised or removed fromsaid source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.Such a nucleotide sequence or segment is commonly referred to as“recombinant.”

Therefore a useful nucleotide sequence, segment or fragment of interestincludes completely synthetic DNA, semi-synthetic DNA, DNA isolated frombiological sources, and DNA derived from introduced RNA. Generally, theintroduced DNA is not originally resident in the plant genotype which isthe recipient of the DNA, but it is within the scope of the invention toisolate a gene from a given plant genotype, and to subsequentlyintroduce multiple copies of the gene into the same genotype, e.g., toenhance production of a given gene product such as a storage protein ora protein that confers tolerance or resistance to water deficit.

The introduced recombinant DNA molecule includes but is not limited to,DNA from plant genes, and non-plant genes such as those from bacteria,yeasts, animals or viruses.

The introduced DNA can include modified genes, portions of genes, orchimeric genes, including genes from the same or different genotype. Theterm “chimeric gene” or “chimeric DNA” is defined as a gene or DNAsequence or segment comprising at least two DNA sequences or segmentsfrom species which do not combine DNA under natural conditions, or whichDNA sequences or segments are positioned or linked in a manner whichdoes not normally occur in the native genome of untransformed plant.

The introduced recombinant DNA molecule used for transformation hereinmay be circular or linear, double-stranded or single-stranded.Generally, the DNA is in the form of chimeric DNA, such as plasmid DNA,that can also contain coding regions flanked by regulatory sequences,which promote the expression of the recombinant DNA present in theresultant plant. Generally, the introduced recombinant DNA molecule willbe relatively small, i.e., less than about 30 kb to minimize anysusceptibility to physical, chemical, or enzymatic degradation which isknown to increase as the size of the nucleotide molecule increases. Asnoted above, the number of proteins, RNA transcripts or mixturesthereof, which is introduced into the plant genome, is preferablypreselected and defined, e.g., from one to about 5-10 such products ofthe introduced DNA may be formed.

Two principal methods for the control of expression are known, viz.:overexpression and underexpression. Overexpression can be achieved byinsertion of one or more than one extra copy of the selected gene. Itis, however, not unknown for plants or their progeny, originallytransformed with one or more than one extra copy of a nucleotidesequence, to exhibit the effects of underexpression as well asoverexpression. For underexpression there are two principle methods,which are commonly referred to in the art as “antisense downregulation”and “sense downregulation” (sense downregulation is also referred to as“cosuppression”). Generically these processes are referred to as “genesilencing”. Both of these methods lead to an inhibition of expression ofthe target gene.

Obtaining sufficient levels of transgene expression in the appropriateplant tissues is an important aspect in the production of geneticallyengineered crops. Expression of heterologous DNA sequences in a planthost is dependent upon the presence of an operably linked promoter thatis functional within the plant host. Choice of the promoter sequencewill determine when and where within the organism the heterologous DNAsequence is expressed.

It is specifically contemplated by the inventors that one couldmutagenize a promoter to potentially improve the utility of the elementsfor the expression of transgenes in plants. The mutagenesis of theseelements can be carried out at random and the mutagenized promotersequences screened for activity in a trial-by-error procedure.Alternatively, particular sequences which provide the promoter withdesirable expression characteristics, or the promoter with expressionenhancement activity, could be identified and these or similar sequencesintroduced into the sequences via mutation. It is further contemplatedthat one could mutagenize these sequences in order to enhance theirexpression of transgenes in a particular species.

The means for mutagenizing a DNA segment encoding a promoter sequence ofthe current invention are well known to those of skill in the art. Asindicated, modifications to promoter or other regulatory element may bemade by random, or site-specific mutagenesis procedures. The promoterand other regulatory element may be modified by altering their structurethrough the addition or deletion of one or more nucleotides from thesequence which encodes the corresponding unmodified sequences.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art, such as, and not limited to, synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular regulatory region. In particular, site-specific mutagenesisis a technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector, which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephages are readily commercially available and their use is generallywell known to those skilled in the art. Double stranded plasmids alsoare routinely employed in site directed mutagenesis, which eliminatesthe step of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the promoter. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as E. colipolymerase I Klenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform or transfect appropriate cells, such as E. coli cells, andcells are selected which include recombinant vectors bearing the mutatedsequence arrangement. Vector DNA can then be isolated from these cellsand used for plant transformation. A genetic selection scheme wasdevised by Kunkel et al. (1987) to enrich for clones incorporatingmutagenic oligonucleotides. Alternatively, the use of PCR withcommercially available thermostable enzymes such as Taq polymerase maybe used to incorporate a mutagenic oligonucleotide primer into anamplified DNA fragment that can then be cloned into an appropriatecloning or expression vector. The PCR-mediated mutagenesis procedures ofTomic et al. (1990) and Upender et al. (1995) provide two examples ofsuch protocols. A PCR employing a thermostable ligase in addition to athermostable polymerase also may be used to incorporate a phosphorylatedmutagenic oligonucleotide into an amplified DNA fragment that may thenbe cloned into an appropriate cloning or expression vector. Themutagenesis procedure described by Michael (1994) provides an example ofone such protocol.

The preparation of sequence variants of the selected promoter-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting, asthere are other ways in which sequence variants of DNA sequences may beobtained. For example, recombinant vectors encoding the desired promotersequence may be treated with mutagenic agents, such as hydroxylamine, toobtain sequence variants.

As used herein; the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing (see, for example, Watson and Rarnstad, 1987). Typically, vectormediated methodologies involve the introduction of the nucleic acidfragment into a DNA or RNA vector, the clonal amplification of thevector, and the recovery of the amplified nucleic acid fragment.Examples of such methodologies are provided by U.S. Pat. No. 4,237,224.A number of template-dependent processes are available to amplify thetarget sequences of interest present in a sample, such methods beingwell known in the art and specifically disclosed herein below.

Where a clone comprising a promoter has been isolated in accordance withthe instant invention, one may wish to delimit the essential promoterregions within the clone. One efficient, targeted means for preparingmutagenizing promoters relies upon the identification of putativeregulatory elements within the promoter sequence. This can be initiatedby comparison with promoter sequences known to be expressed in similartissue-specific or developmentally unique manner. Sequences, which areshared among promoters with similar expression patterns, are likelycandidates for the binding of transcription factors and are thus likelyelements that confer expression patterns. Confirmation of these putativeregulatory elements can be achieved by deletion analysis of eachputative regulatory region followed by functional analysis of eachdeletion construct by assay of a reporter gene, which is functionallyattached to each construct. As such, once a starting promoter sequenceis provided, any of a number of different deletion mutants of thestarting promoter could be readily prepared.

Functionally equivalent fragments of a transcription regulatingnucleotide sequence of the invention can also be obtained by removing ordeleting non-essential sequences without deleting the essential one.Narrowing the transcription regulating nucleotide sequence to itsessential, transcription mediating elements can be realized in vitro bytrial-and-arrow deletion mutations, or in silico using promoter elementsearch routines. Regions essential for promoter activity oftendemonstrate clusters of certain, known promoter elements. Such analysiscan be performed using available computer algorithms such as PLACE(“Plant Cis-acting Regulatory DNA Elements”; Higo 1999), the BIOBASEdatabase “Transfac” (Biologische Datenbanken GmbH, Braunschweig;Wingender 2001) or the database PlantCARE (Lescot 2002).

Preferably, functional equivalent fragments of one of the transcriptionregulating nucleotide sequences of the invention comprises at least 100base pairs, preferably, at least 200 base pairs, more preferably atleast 500 base pairs of a transcription regulating nucleotide sequenceas described by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28, 31, 32, 33, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74, 75, 76, 77, 80, 81, 84, 85,86, 87, 148, 149, 150, 151, 152, 153, 154, 155, 158, 159, 160, 161, 162,163, 164, 165, 168, 169, 170, 171, 172, 173, 174, 175, 178, 179, 180,181, 182, 183, 184, or 185. More preferably this fragment is startingfrom the 3′-end of the indicated sequences.

Especially preferred are equivalent fragments of transcriptionregulating nucleotide sequences, which are obtained by deleting theregion encoding the 5′-untranslated region of the mRNA, thus onlyproviding the (untranscribed) promoter region. The 5′-untranslatedregion can be easily determined by methods known in the art (such as5′-RACE analysis). Accordingly, some of the transcription regulatingnucleotide sequences of the invention are equivalent fragments of othersequences (see Table 2 below).

TABLE 2 Relationship of transcription regulating nucleotide sequences ofthe invention Transcription regulating sequence Equivalent sequenceEquivalent fragment SEQ ID NO: 9 (3954 bp) SEQ ID NO: 10 (3962 bp) SEQID NO: 1 (980 bp) SEQ ID NO: 2 (994 bp) SEQ ID NO: 3 (686 bp) SEQ ID NO:4 (698 bp) SEQ ID NO: 5 (2051 bp) SEQ ID NO: 6 (2066 bp) SEQ ID NO: 7(1757 bp) SEQ ID NO: 8 (1770 bp) SEQ ID NO: 11 (3660 bp) SEQ ID NO: 12(3666 bp) SEQ ID NO: 19 (3892 bp) SEQ ID NO: 20 (3083 bp) SEQ ID NO: 15(2254 bp) SEQ ID NO: 16 (2259 bp) SEQ ID NO: 17 (1446 bp) SEQ ID NO: 18(1450 bp) SEQ ID NO: 27 (2352 bp) SEQ ID NO: 28 (2168 bp) SEQ ID NO: 23(1026 bp) SEQ ID NO: 24 (1030 bp) SEQ ID NO: 25 (844 bp) SEQ ID NO: 26(846 bp) SEQ ID NO: 33 (1441 bp) — SEQ ID NO: 31 (1201 bp) SEQ ID NO: 32(1216 bp) SEQ ID NO: 36 (1587 bp) SEQ ID NO: 37 (1602 bp) SEQ ID NO: 38(1130 bp) SEQ ID NO: 39 (1145 bp) SEQ ID NO: 40 (1169 bp) SEQ ID NO: 41(1191 bp) SEQ ID NO: 42 (1130 bp) SEQ ID NO: 43 (1145 bp) SEQ ID NO: 44(1120 bp) SEQ ID NO: 45 (1135 bp) SEQ ID NO: 46 (663 bp) SEQ ID NO: 47(678 bp) SEQ ID NO: 54 (2012 bp) SEQ ID NO: 55 (2033 bp) SEQ ID NO: 50(1069 bp) SEQ ID NO: 51 (1088 bp) SEQ ID NO: 52 (1034 bp) SEQ ID NO: 53(1053 bp) SEQ ID NO: 56 (1977 bp) SEQ ID NO: 57 (1998 bp) SEQ ID NO: 68(2996 bp) SEQ ID NO: 69 (3008 bp) SEQ ID NO: 60 (949 bp) SEQ ID NO: 61(962 bp) SEQ ID NO: 62 (768 bp) SEQ ID NO: 63 (779 bp) SEQ ID NO: 64(2040 bp) SEQ ID NO: 65 (2053 bp) SEQ ID NO: 66 (1859 bp) SEQ ID NO: 67(1870 bp) SEQ ID NO: 70 (2815 bp) SEQ ID NO: 71 (2825 bp) SEQ ID NO: 74(1564 bp) SEQ ID NO: 75 (1578 bp) SEQ ID NO: 76 (1404 bp) SEQ ID NO: 77(1418 bp) SEQ ID NO: 80 (524 bp) SEQ ID NO: 81(539 bp) — SEQ ID NO: 84(1988 bp) SEQ ID NO: 85 (2010 bp) SEQ ID NO: 86 (1776 bp) SEQ ID NO: 87(1800 bp) SEQ ID NO: 152 (1845 bp) SEQ ID NO: 153 (1854 bp) SEQ ID NO:148 (1106 bp) SEQ ID NO: 149 (1115 bp) SEQ ID NO: 150 (923 bp) SEQ IDNO: 151 (930 bp) SEQ ID NO: 154 (1662 bp) SEQ ID NO: 155 (1669 bp) SEQID NO: 162 (2017 bp) SEQ ID NO: 163 (2004 bp) SEQ ID NO: 158 (1017 bp)SEQ ID NO: 159 (1008 bp) SEQ ID NO: 160 (894 bp) SEQ ID NO: 161 (886 bp)SEQ ID NO: 164 (1894 bp) SEQ ID NO: 165 (1882 bp) SEQ ID NO: 172 (3232bp) SEQ ID NO: 173 (3269 bp) SEQ ID NO: 168 (1300 bp) SEQ ID NO: 169(1337 bp) SEQ ID NO: 170 (1190 bp) SEQ ID NO: 171 (1226 bp) SEQ ID NO:174 (3123 bp) SEQ ID NO: 175 (3158 bp) SEQ ID NO: 182 (3019 bp) SEQ IDNO: 183 (3006 bp) SEQ ID NO: 178 (1504 bp) SEQ ID NO: 179 (1492 bp) SEQID NO: 180 (1450 bp) SEQ ID NO: 181 (1436 bp) SEQ ID NO: 184 (2965 bp)SEQ ID NO: 185 (2950 bp)

As indicated above, deletion mutants, deletion mutants of the promoterof the invention also could be randomly prepared and then assayed. Withthis strategy, a series of constructs are prepared, each containing adifferent portion of the clone (a subclone), and these constructs arethen screened for activity. A suitable means for screening for activityis to attach a deleted promoter or intron construct, which contains adeleted segment to a selectable or screenable marker, and to isolateonly those cells expressing the marker gene. In this way, a number ofdifferent, deleted promoter constructs are identified which still retainthe desired, or even enhanced, activity. The smallest segment, which isrequired for activity, is thereby identified through comparison of theselected constructs. This segment may then be used for the constructionof vectors for the expression of exogenous genes.

An expression cassette of the invention may comprise further regulatoryelements. The term in this context is to be understood in the a broadmeaning comprising all sequences which may influence construction orfunction of the expression cassette. Regulatory elements may for examplemodify transcription and/or translation in prokaryotic or eukaryoticorganism. In an preferred embodiment the expression cassette of theinvention comprised downstream (in 3′-direction) of the nucleic acidsequence to be expressed a transcription termination sequenceand—optionally additional regulatory elements—each operably liked to thenucleic acid sequence to be expressed (or the transcription regulatingnucleotide sequence).

Additional regulatory elements may comprise additional promoter, minimalpromoters, or promoter elements, which may modify the expressionregulating properties. For example the expression may be made dependingon certain stress factors such water stress, abscisin (Lam 1991) or heatstress (Schoffl 1989). Furthermore additional promoters or promoterelements may be employed, which may realize expression in otherorganisms (such as E. coli or Agrobacterium). Such regulatory elementscan be found in the promoter sequences or bacteria such as amy and SPO2or in the promoter sequences of yeast or fungal promoters (such as ADC1,MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, and ADH).

Furthermore, it is contemplated that promoters combining elements frommore than one promoter may be useful. For example, U.S. Pat. No.5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with ahistone promoter. Thus, the elements from the promoters disclosed hereinmay be combined with elements from other promoters. Promoters, which areuseful for plant transgene expression include those that are inducible,viral, synthetic, constitutive (Odell 1985), temporally regulated,spatially regulated, tissue-specific, and spatial-temporally regulated.

Where expression in specific tissues or organs is desired,tissue-specific promoters may be used. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory elements of choice. Where continuous expression isdesired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

A variety of 5′ and 3′ transcriptional regulatory sequences areavailable for use in the present invention. Transcriptional terminatorsare responsible for the termination of transcription and correct mRNApolyadenylation. The 3′ nontranslated regulatory DNA sequence preferablyincludes from about 50 to about 1,000, more preferably about 100 toabout 1,000, nucleotide base pairs and contains plant transcriptionaland translational termination sequences. Appropriate transcriptionalterminators and those which are known to function in plants include theCaMV 35S terminator, the tml terminator, the nopaline synthaseterminator, the pea rbcS E9 terminator, the terminator for the T7transcript from the octopine synthase gene of Agrobacterium tumefaciens,and the 3′ end of the protease inhibitor I or II genes from potato ortomato, although other 3′ elements known to those of skill in the artcan also be employed. Alternatively, one also could use a gamma coixin,oleosin 3 or other terminator from the genus Coix.

Preferred 3′ elements include those from the nopaline synthase gene ofAgrobacterium tumefaciens (Bevan 1983), the terminator for the T7transcript from the octopine synthase gene of Agrobacterium tumefaciens,and the 3′ end of the protease inhibitor I or II genes from potato ortomato.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose, which include sequences, predicted to direct optimum expressionof the attached gene, i.e., to include a preferred consensus leadersequence, which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants will be most preferred.

Preferred regulatory elements also include the 5′-untranslated region,introns and the 3′-untranslated region of genes. Such sequences thathave been found to enhance gene expression in transgenic plants includeintron sequences (e.g., from Adh1, bronze1, actin1, actin 2 (WO00/760067), or the sucrose synthase intron; see: The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)) andviral leader sequences (e.g., from TMV, MCMV and AMV; Gallie 1987). Forexample, a number of non-translated leader sequences derived fromviruses are known to enhance expression. Specifically, leader sequencesfrom Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV),and Alfalfa Mosaic Virus (AMV) have been shown to be effective inenhancing expression (e.g., Gallie 1987; Skuzeski 1990). Other leadersknown in the art include but are not limited to: Picornavirus leaders,for example, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein 1989); Potyvirus leaders, for example, TEV leader (TobaccoEtch Virus); MDMV leader (Maize Dwarf Mosaic Virus); Humanimmunoglobulin heavy-chain binding protein (BiP) leader, (Macejak 1991);Untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4), (Jobling 1987; Tobacco mosaic virus leader (TMV), (Gallie1989; and Maize Chlorotic Mottle Virus leader (MCMV) (Lommel 1991. Seealso, Della-Cioppa 1987. Regulatory elements such as Adh intron 1(Callis 1987), sucrose synthase intron (Vasil 1989) or TMV omega element(Gallie 1989), may further be included where desired. Especiallypreferred are the 5′-untranslated region, introns and the3+-untranslated region from the genes described by the GenBankArabidopsis thaliana genome locii At4g12910, At1g66250, At4g00820,At2g36640, At2g34200, At3g1180, At4g00360, At2g48030, At2g38590,At1g23000, At3g15510, At3g50870, At4g00220, or At4g26320, or offunctional equivalent thereof.

Additional preferred regulatory elements are enhancer sequences orpolyadenylation sequences. Preferred polyadenylation sequences are thosefrom plant genes or Agrobacterium T-DNA genes (such as for example theterminator sequences of the OCS (octopine synthase) or NOS (nopalinesynthase) genes).

Examples of enhancers include elements from the CaMV 35S promoter,octopine synthase genes (Ellis el al., 1987), the rice actin I gene, themaize alcohol dehydrogenase gene (Callis 1987), the maize shrunken Igene (Vasil 1989), TMV Omega element (Gallie 1989) and promoters fromnon-plant eukaryotes (e.g. yeast; Ma 1988). Vectors for use inaccordance with the present invention may be constructed to include theocs enhancer element. This element was first identified as a 16 bppalindromic enhancer from the octopine synthase (ocs) gene of ultilane(Ellis 1987), and is present in at least 10 other promoters (Bouchez1989). The use of an enhancer element, such as the ocs elements andparticularly multiple copies of the element, will act to increase thelevel of transcription from adjacent promoters when applied in thecontext of plant transformation.

An expression cassette of the invention (or a vector derived therefrom)may comprise additional functional elements, which are to be understoodin the broad sense as all elements which influence construction,propagation, or function of an expression cassette or a vector or atransgenic organism comprising them. Such functional elements mayinclude origin of replications (to allow replication in bacteria; forthe ORI of pBR322 or the P15A ori; Sambrook 1989), or elements requiredfor Agrobacterium T-DNA transfer (such as for example the left and/orrights border of the T-DNA).

Ultimately, the most desirable DNA segments for introduction into, forexample, a dicot genome, may be homologous genes or gene families whichencode a desired trait (e.g., increased yield per acre) and which areintroduced under the control of novel promoters or enhancers, etc., orperhaps even homologous or tissue specific (e.g., root-, collar/sheath-,whorl-, stalk-, earshank-, kernel- or leaf-specific) promoters orcontrol elements. Indeed, it is envisioned that a particular use of thepresent invention will be the expression of a gene in aseed-preferential or seed-specific manner.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit or signal peptide willtransport the protein to a particular intracellular or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicideresistance gene, such as the EPSPS gene, to a particular organelle suchas the chloroplast rather than to the cytoplasm. This is exemplified bythe use of the rbcs transit peptide which confers plastid-specifictargeting of proteins. In addition, it is proposed that it may bedesirable to target certain genes responsible for male sterility to themitochondria, or to target certain genes for resistance tophytopathogenic organisms to the extracellular spaces, or to targetproteins to the vacuole.

By facilitating the transport of the protein into compartments insideand outside the cell, these sequences may increase the accumulation ofgene product protecting them from proteolytic degradation. Thesesequences also allow for additional mRNA sequences from highly expressedgenes to be attached to the coding sequence of the genes. Since mRNAbeing translated by ribosomes is more stable than naked mRNA, thepresence of translatable mRNA in front of the gene may increase theoverall stability of the mRNA transcript from the gene and therebyincrease synthesis of the gene product. Since transit and signalsequences are usually post-translationally removed from the initialtranslation product, the use of these sequences allows for the additionof extra translated sequences that may not appear on the finalpolypeptide. Targeting of certain proteins may be desirable in order toenhance the stability of the protein (U.S. Pat. No. 5,545,818).

It may be useful to target DNA itself within a cell. For example, it maybe useful to target introduced DNA to the nucleus as this may increasethe frequency of transformation. Within the nucleus itself it would beuseful to target a gene in order to achieve site-specific integration.For example, it would be useful to have a gene introduced throughtransformation replace an existing gene in the cell. Other elementsinclude those that can be regulated by endogenous or exogenous agents,e.g., by zinc finger proteins, including naturally occurring zinc fingerproteins or chimeric zinc finger proteins (see, e.g., U.S. Pat. No.5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) or myb-liketranscription factors. For example, a chimeric zinc finger protein mayinclude amino acid sequences, which bind to a specific DNA sequence (thezinc finger) and amino acid sequences that activate (e.g., GAL 4sequences) or repress the transcription of the sequences linked to thespecific DNA sequence.

It is one of the objects of the present invention to provide recombinantDNA molecules comprising a nucleotide sequence according to theinvention operably linked to a nucleotide segment of interest.

A nucleotide segment of interest is reflective of the commercial marketsand interests of those involved in the development of the crop. Cropsand markets of interest changes, and as developing nations open up worldmarkets, new crops and technologies will also emerge. In addition, asthe understanding of agronomic traits and characteristics such as yieldand heterosis increase, the choice of genes for transformation willchange accordingly. General categories of nucleotides of interestinclude, for example, genes involved in information, such as zincfingers, those involved in communication, such as kinases, and thoseinvolved in housekeeping, such as heat shock proteins. More specificcategories of transgenes, for example, include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, sterility, grain characteristics, and commercial products.Genes of interest include, generally, those involved in starch, oil,carbohydrate, or nutrient metabolism, as well as those affecting kernelsize, sucrose loading, zinc finger proteins, see, e.g., U.S. Pat. No.5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO98/53058; WO 00/23464; WO 95/19431; and WO 98/54311, and the like.

One skilled in the art recognizes that the expression level andregulation of a transgene in a plant can vary significantly from line toline. Thus, one has to test several lines to find one with the desiredexpression level and regulation. Once a line is identified with thedesired regulation specificity of a chimeric Cre transgene, it can becrossed with lines carrying different inactive replicons or inactivetransgene for activation.

Other sequences which may be linked to the gene of interest, whichencodes a polypeptide, are those which can target to a specificorganelle, e.g., to the mitochondria, nucleus, or plastid, within theplant cell. Targeting can be achieved by providing the polypeptide withan appropriate targeting peptide sequence, such as a secretory signalpeptide (for secretion or cell wall or membrane targeting, a plastidtransit peptide, a chloroplast transit peptide, e.g., the chlorophylla/b binding protein, a mitochondrial target peptide, a vacuole targetingpeptide, or a nuclear targeting peptide, and the like. For example, thesmall subunit of ribulose bisphosphate carboxylase transit peptide, theEPSPS transit peptide or the dihydrodipicolinic acid synthase transitpeptide may be used, for examples of plastid organelle targetingsequences (see WO 00/12732). Plastids are a class of plant organellesderived from proplastids and include chloroplasts, leucoplasts,amyloplasts, and chromoplasts. The plastids are major sites ofbiosynthesis in plants. In addition to photosynthesis in thechloroplast, plastids are also sites of lipid biosynthesis, nitratereduction to ammonium, and starch storage. And while plastids containtheir own circular, genome, most of the proteins localized to theplastids are encoded by the nuclear genome and are imported into theorganelle from the cytoplasm.

Transgenes used with the present invention will often be genes thatdirect the expression of a particular protein or polypeptide product,but they may also be nonexpressible DNA segments, e.g., transposons suchas Ds that do no direct their own transposition. As used herein, an“expressible gene” is any gene that is capable of being transcribed intoRNA (e.g., mRNA, antisense RNA, etc.) or translated into a protein,expressed as a trait of interest, or the like, etc., and is not limitedto selectable, screenable or non-selectable marker genes. The inventionalso contemplates that, where both an expressible gene that is notnecessarily a marker gene is employed in combination with a marker gene,one may employ the separate genes on either the same or different DNAsegments for transformation. In the latter case, the different vectorsare delivered concurrently to recipient cells to maximizecotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch properties; oilquantity and quality; and the like. One may desire to incorporate one ormore genes conferring any such desirable trait or traits, such as, forexample, a gene or genes encoding pathogen resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, or colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar in combinationwith either of the above genes. Of course, any two or more transgenes ofany description, such as those conferring herbicide, insect, disease(viral, bacterial, fungal, nematode) or drought resistance, malesterility, drydown, standability, prolificacy, starch properties, oilquantity and quality, or those increasing yield or nutritional qualitymay be employed as desired.

1. Exemplary Transgenes

1.1. Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP Synthaseenzymes. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

1.2 Insect Resistance

An important aspect of the present invention concerns the introductionof insect resistance-conferring genes into plants. Potential insectresistance genes, which can be introduced, include Bacillusthuringiensis crystal toxin genes or Bt genes (Watrud 1985). Bt genesmay provide resistance to lepidopteran or coleopteran pests such asEuropean Corn Borer (ECB) and corn rootworm (CRW). Preferred Bt toxingenes for use in such embodiments include the CrylA(b) and CrylA(c)genes. Endotoxin genes from other species of B. thuringiensis, whichaffect insect growth or development, may also be employed in thisregard. Protease inhibitors may also provide insect resistance (Johnson1989), and will thus have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered by the present inventors to produce synergisticinsecticidal activity. Other genes, which encode inhibitors of theinsects' digestive system, or those that encode enzymes or co-factorsthat facilitate the production of inhibitors, may also be useful.Cystatin and amylase inhibitors, such as those from wheat and barley,may exemplify this group.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins, which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock 1990; Czapla & Lang, 1990). Lectingenes contemplated to be useful include, for example, barley and wheatgerm agglutinin (WGA) and rice lectins (Gatehouse 1984), with WGA beingpreferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated, that theexpression of juvenile hormone esterase, directed towards specificinsect pests, may also result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock 1990).

Transgenic plants expressing genes, which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant maize plants. Genesthat code for activities that affect insect molting, such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase, alsofall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsare also encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

The present invention also provides methods and compositions by which toachieve qualitative or quantitative changes in plant secondarymetabolites. One example concerns transforming plants to produce DIMBOAwhich, it is contemplated, will confer resistance to European cornborer, rootworm and several other maize insect pests. Candidate genesthat are particularly considered for use in this regard include thosegenes at the bx locus known to be involved in the synthetic DIMBOApathway (Dunn 1981). The introduction of genes that can regulate theproduction of maysin, and genes involved in the production of dhurrin insorghum, is also contemplated to be of use in facilitating resistance toearworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn rootworm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson & Guss, 1972).

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder 1987) which may be used as a rootworm deterrent; genesencoding avermectin (Campbell 1989; Ikeda 1987) which may proveparticularly useful as a corn rootworm deterrent; ribosome inactivatingprotein genes; and even genes that regulate plant structures. Transgenicmaize including anti-insect antibody genes and genes that code forenzymes that can covert a non-toxic insecticide (pro-insecticide)applied to the outside of the plant into an insecticide inside the plantare also contemplated.

1.3 Environment or Stress Resistance

Improvement of a plant's ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, canalso be effected through expression of heterologous, or overexpressionof homologous genes. Benefits may be realized in terms of increasedresistance to freezing temperatures through the introduction of an“antifreeze” protein such as that of the Winter Flounder (Cutler 1989)or synthetic gene derivatives thereof. Improved chilling tolerance mayalso be conferred through increased expression of glycerol-3-phosphateacetyltransferase in chloroplasts (Murata 1992; Wolter 1992). Resistanceto oxidative stress (often exacerbated by conditions such as chillingtemperatures in combination with high light intensities) can beconferred by expression of superoxide dismutase (Gupta 1993), and may beimproved by glutathione reductase (Bowler 1992). Such strategies mayallow for tolerance to freezing in newly emerged fields as well asextending later maturity higher yielding varieties to earlier relativematurity zones.

Expression of novel genes that favorably effect plant water content,total water potential, osmotic potential, and turgor can enhance theability of the plant to tolerate drought. As used herein, the terms“drought resistance” and “drought tolerance” are used to refer to aplants increased resistance or tolerance to stress induced by areduction in water availability, as compared to normal circumstances,and the ability of the plant to function and survive in lower-waterenvironments, and perform in a relatively superior manner. In thisaspect of the invention it is proposed, for example, that the expressionof a gene encoding the biosynthesis of osmotically active solutes canimpart protection against drought. Within this class of genes are DNAsencoding mannitol dehydrogenase (Lee and Saier, 1982) andtrehalose-6-phosphate synthase (Kaasen 1992). Through the subsequentaction of native phosphatases in the cell or by the introduction andcoexpression of a specific phosphatase, these introduced genes willresult in the accumulation of either mannitol or trehalose,respectively, both of which have been well documented as protectivecompounds able to mitigate the effects of stress. Mannitol accumulationin transgenic tobacco has been verified and preliminary results indicatethat plants expressing high levels of this metabolite are able totolerate an applied osmotic stress (Tarczynski 1992).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g. alanopine or propionic acid) or membrane integrity (e.g.,alanopine) has been documented (Loomis 1989), and therefore expressionof gene encoding the biosynthesis of these compounds can confer droughtresistance in a manner similar to or complimentary to mannitol. Otherexamples of naturally occurring metabolites that are osmotically activeand/or provide some direct protective effect during drought and/ordesiccation include sugars and sugar derivatives such as fructose,erythritol (Coxson 1992), sorbitol, dulcitol (Karsten 1992),glucosylglycerol (Reed 1984; Erdmann 1992), sucrose, stachyose (Koster &Leopold 1988; Blackman 1992), ononitol and pinitol (Vernon & Bohnert1992), and raffinose (Bernal-Lugo & Leopold 1992). Other osmoticallyactive solutes, which are not sugars, include, but are not limited to,proline and glycine-betaine (Wyn-Jones and Storey, 1981). Continuedcanopy growth and increased reproductive fitness during times of stresscan be augmented by introduction and expression of genes such as thosecontrolling the osmotically active compounds discussed above and othersuch compounds, as represented in one exemplary embodiment by the enzymemyoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins may alsoincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure 1989). Allthree classes of these proteins have been demonstrated in maturing(i.e., desiccating) seeds. Within these 3 types of proteins, the Type-II(dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (e.g. Mundy and Chua,1988; Piatkowski 1990; Yamaguchi-Shinozaki 1992). Recently, expressionof a Type-III LEA (HVA-1) in tobacco was found to influence plantheight, maturity and drought tolerance (Fitzpatrick, 1993). Expressionof structural genes from all three groups may therefore confer droughttolerance. Other types of proteins induced during water stress includethiol proteases, aldolases and transmembrane transporters (Guerrero1990), which may confer various protective and/or repair-type functionsduring drought stress. The expression of a gene that effects lipidbiosynthesis and hence membrane composition can also be useful inconferring drought resistance on the plant.

Many genes that improve drought resistance have complementary modes ofaction. Thus, combinations of these genes might have additive and/orsynergistic effects in improving drought resistance in maize. Many ofthese genes also improve freezing tolerance (or resistance); thephysical stresses incurred during freezing and drought are similar innature and may be mitigated in similar fashion. Benefit may be conferredvia constitutive expression or tissue-specific of these genes, but thepreferred means of expressing these novel genes may be through the useof a turgor-induced promoter (such as the promoters for theturgor-induced genes described in Guerrero et al. 1990 and Shagan 1993).Spatial and temporal expression patterns of these genes may enable maizeto better withstand stress.

Expression of genes that are involved with specific morphological traitsthat allow for increased water extractions from drying soil would be ofbenefit. For example, introduction and expression of genes that alterroot characteristics may enhance water uptake. Expression of genes thatenhance reproductive fitness during times of stress would be ofsignificant value. For example, expression of DNAs that improve thesynchrony of pollen shed and receptiveness of the female flower parts,i.e., silks, would be of benefit. In addition, expression of genes thatminimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value. Regulation ofcytokinin levels in monocots, such as maize, by introduction andexpression of an isopentenyl transferase gene with appropriateregulatory sequences can improve monocot stress resistance and yield(Gan 1995).

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

Improved protection of the plant to abiotic stress factors such asdrought, heat or chill, can also be achieved—for example—byoverexpressing antifreeze polypeptides from Myoxocephalus Scorpius (WO00/00512), Myoxocephalus octodecemspinosus, the Arabidopsis thalianatranscription activator CBF1, glutamate dehydrogenases (WO 97/12983, WO98/11240), calcium-dependent protein kinase genes (WO 98/26045),calcineurins (WO 99/05902), casein kinase from yeast (WO 02/052012),farnesyltransferases (WO 99/06580; Pei Z M et al. (1998) Science282:287-290), ferritin (Deak M et al. (1999) Nature Biotechnology17:192-196), oxalate oxidase (WO 99/04013; Dunwell J M (1998) BiotechnGenet Eng Rev 15:1-32), DREB1A factor (“dehydration response element B1A”; Kasuga M et al. (1999) Nature Biotech 17:276-286), genes ofmannitol or trehalose synthesis such as trehalose-phosphate synthase ortrehalose-phosphate phosphatase (WO 97/42326) or by inhibiting genessuch as trehalase (WO 97/50561).

1.4 Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants period. It is possible toproduce resistance to diseases caused, by viruses, bacteria, fungi, rootpathogens, insects and nematodes. It is also contemplated that controlof mycotoxin producing organisms may be realized through expression ofintroduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo1988, Hemenway 1988, Abel 1986). It is contemplated that expression ofantisense genes targeted at essential viral functions may impartresistance to said virus. For example, an antisense gene targeted at thegene responsible for replication of viral nucleic acid may inhibit saidreplication and lead to resistance to the virus. It is believed thatinterference with other viral functions through the use of antisensegenes may also increase resistance to viruses. Further it is proposedthat it may be possible to achieve resistance to viruses through otherapproaches, including, but not limited to the use of satellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences, which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in plants may beuseful in conferring resistance to bacterial disease. These genes areinduced following pathogen attack on a host plant and have been dividedinto at least five classes of proteins (Bol 1990). Included amongst thePR proteins are beta-1,3-glucanases, chitinases, and osmotin and otherproteins that are believed to function in plant resistance to diseaseorganisms. Other genes have been identified that have antifungalproperties, e.g., UDA (stinging nettle lectin) and hevein (Broakgert1989; Barkai-Golan 1978). It is known that certain plant diseases arecaused by the production of phytotoxins. Resistance to these diseasescould be achieved through expression of a novel gene that encodes anenzyme capable of degrading or otherwise inactivating the phytotoxin.Expression novel genes that alter the interactions between the hostplant and pathogen may be useful in reducing the ability the diseaseorganism to invade the tissues of the host plant, e.g., an increase inthe waxiness of the leaf cuticle or other morphological characteristics.

Plant parasitic nematodes are a cause of disease in many plants. It isproposed that it would be possible to make the plant resistant to theseorganisms through the expression of novel genes. It is anticipated thatcontrol of nematode infestations would be accomplished by altering theability of the nematode to recognize or attach to a host plant and/orenabling the plant to produce nematicidal compounds, including but notlimited to proteins.

Furthermore, a resistance to fungi, insects, nematodes and diseases, canbe achieved by by targeted accumulation of certain metabolites orproteins. Such proteins include but are not limited to glucosinolates(defense against herbivores), chitinases or glucanases and other enzymeswhich destroy the cell wall of parasites, ribosome-inactivating proteins(RIPs) and other proteins of the plant resistance and stress reaction asare induced when plants are wounded or attacked by microbes, orchemically, by, for example, salicylic acid, jasmonic acid or ethylene,or lysozymes from nonplant sources such as, for example, T4-lysozyme orlysozyme from a variety of mammals, insecticidal proteins such asBacillus thuringiensis endotoxin, a-amylase inhibitor or proteaseinhibitors (cowpea trypsin inhibitor), lectins such as wheatgermagglutinin, RNAses or ribozymes. Further examples are nucleic acidswhich encode the Trichoderma harzianum chit42 endochitinase (GenBankAcc. No.: S78423) or the N-hydroxylating, multi-functional cytochromeP-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U32624),or functional equivalents of these. The accumulation of glucosinolatesas protection from pests (Rask L et al. (2000) Plant Mol Biol 42:93-113;Menard R et al. (1999) Phytochemistry 52:29-35), the expression ofBacillus thuringiensis endotoxins (Vaeck et al. (1987) Nature 328:33-37)or the protection against attack by fungi, by expression of chitinases,for example from beans (Broglie et al. (1991) Science 254:1194-1197), isadvantageous. Resistance to pests such as, for example, the rice pestNilaparvata lugens in rice plants can be achieved by expressing thesnowdrop (Galanthus nivalis) lectin agglutinin (Rao et al. (1998) PlantJ 15(4):469-77).The expression of synthetic crylA(b) and crylA(c) genes,which encode lepidoptera-specific Bacillus thuringiensis D-endotoxinscan bring about a resistance to insect pests in various plants (Goyal RK et al. (2000) Crop Protection 19(5):307-312). Further target geneswhich are suitable for pathogen defense comprise“polygalacturonase-inhibiting protein” (PGIP), thaumatine, invertase andantimicrobial peptides such as lactoferrin (Lee T J et al. (2002) J AmerSoc Horticult Sci 127(2):158-164).

1.5 Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with plants is a significant factor in rendering the grainnot useful. These fungal organisms do not cause disease symptoms and/orinterfere with the growth of the plant, but they produce chemicals(mycotoxins) that are toxic to animals. Inhibition of the growth ofthese fungi would reduce the synthesis of these toxic substances and,therefore, reduce grain losses due to mycotoxin contamination. Novelgenes may be introduced into plants that would inhibit synthesis of themycotoxin without interfering with fungal growth. Expression of a novelgene, which encodes an enzyme capable of rendering the mycotoxinnontoxic, would be useful in order to achieve reduced mycotoxincontamination of grain. The result of any of the above mechanisms wouldbe a reduced presence of mycotoxins on grain.

1.6 Grain Composition or Quality

Genes may be introduced into plants, particularly commercially importantcereals such as maize, wheat or rice, to improve the grain for which thecereal is primarily grown. A wide range of novel transgenic plantsproduced in this manner may be envisioned depending on the particularend use of the grain.

For example, the largest use of maize grain is for feed or food.Introduction of genes that alter the composition of the grain maygreatly enhance the feed or food value. The primary components of maizegrain are starch, protein, and oil. Each of these primary components ofmaize grain may be improved by altering its level or composition.Several examples may be mentioned for illustrative purposes but in noway provide an exhaustive list of possibilities.

The protein of many cereal grains is suboptimal for feed and foodpurposes especially when fed to pigs, poultry, and humans. The proteinis deficient in several amino acids that are essential in the diet ofthese species, requiring the addition of supplements to the grain.Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after the grain is supplemented with other inputsfor feed formulations. For example, when the grain is supplemented withsoybean meal to meet lysine requirements, methionine becomes limiting.The levels of these essential amino acids in seeds and grain may beelevated by mechanisms which include, but are not limited to, theintroduction of genes to increase the biosynthesis of the amino acids,decrease the degradation of the amino acids, increase the storage of theamino acids in proteins, or increase transport of the amino acids to theseeds or grain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway that are normally regulated by levels ofthe amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyse steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.DNA may be introduced that decreases the expression of members of thezein family of storage proteins. This DNA may encode ribozymes orantisense sequences directed to impairing expression of zein proteins orexpression of regulators of zein expression such as the opaque-2 geneproduct. The protein composition of the grain may be modified throughthe phenomenon of cosuppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring 1991).Additionally, the introduced DNA may encode enzymes, which degradezeines. The decreases in zein expression that are achieved may beaccompanied by increases in proteins with more desirable amino acidcomposition or increases in other major seed constituents such asstarch. Alternatively, a chimeric gene may be introduced that comprisesa coding sequence for a native protein of adequate amino acidcomposition such as for one of the globulin proteins or 10 kD zein ofmaize and a promoter or other regulatory sequence designed to elevateexpression of said protein. The coding sequence of said gene may includeadditional or replacement codons for essential amino acids. Further, acoding sequence obtained from another species, or, a partially orcompletely synthetic sequence encoding a completely unique peptidesequence designed to enhance the amino acid composition of the seed maybe employed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable energy content and density of the seeds for uses in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, beta-ketoacyl-ACP synthase,plus other wellknown fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Additional examplesinclude 2-acetyltransferase, oleosin pyruvate dehydrogenase complex,acetyl CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylaseand genes of the carnitine-CoA-acetyl-CoA shuttles. It is anticipatedthat expression of genes related to oil biosynthesis will be targeted tothe plastid, using a plastid transit peptide sequence and preferablyexpressed in the seed embryo. Genes may be introduced that alter thebalance of fatty acids present in the oil providing a more healthful ornutritive feedstuff. The introduced DNA may also encode sequences thatblock expression of enzymes involved in fatty acid biosynthesis,altering the proportions of fatty acids present in the grain such asdescribed below.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA, which blocks or eliminates steps in pigment production pathways.

Feed or food comprising some cereal grains possesses insufficientquantities of vitamins and must be supplemented to provide adequatenutritive value. Introduction of genes that enhance vitamin biosynthesisin seeds may be envisioned including, for example, vitamins A, E, B₁₂,choline, and the like. For example, maize grain also does not possesssufficient mineral content for optimal nutritive value. Genes thataffect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase, whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of cereals for feed and foodpurposes might be described. The improvements may not even necessarilyinvolve the grain, but may, for example, improve the value of the grainfor silage. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.

In addition to direct improvements in feed or food value, genes may alsobe introduced which improve the processing of grain and improve thevalue of the products resulting from the processing. The primary methodof processing certain grains such as maize is via wetmilling. Maize maybe improved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, Theological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs may also be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Furthermore, it may be advisable tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moieties of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups, which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn and other grains, the valueof which may be improved by introduction and expression of genes. Thequantity of oil that can be extracted by wetmilling may be elevated byapproaches as described for feed and food above. Oil properties may alsobe altered to improve its performance in the production and use ofcooking oil, shortenings, lubricants or other oil-derived products orimprovement of its health attributes when used in the food-relatedapplications. Novel fatty acids may also be synthesized which uponextraction can serve as starting materials for chemical syntheses. Thechanges in oil properties may be achieved by altering the type, level,or lipid arrangement of the fatty acids present in the oil. This in turnmay be accomplished by the addition of genes that encode enzymes thatcatalyze the synthesis of novel fatty acids and the lipids possessingthem or by increasing levels of native fatty acids while possiblyreducing levels of precursors. Alternatively DNA sequences may beintroduced which slow or block steps in fatty acid biosynthesisresulting in the increase in precursor fatty acid intermediates. Genesthat might be added include desaturases, epoxidases, hydratases,dehydratases, and other enzymes that catalyze reactions involving fattyacid intermediates. Representative examples of catalytic steps thatmight be blocked include the desaturations from stearic to oleic acidand oleic to linolenic acid resulting in the respective accumulations ofstearic and oleic acids.

Improvements in the other major cereal wetmilling products, gluten mealand gluten feed, may also be achieved by the introduction of genes toobtain novel plants. Representative possibilities include but are notlimited to those described above for improvement of food and feed value.

In addition it may further be considered that the plant be used for theproduction or manufacturing of useful biological compounds that wereeither not produced at all, or not produced at the same level, in theplant previously. The novel plants producing these compounds are madepossible by the introduction and expression of genes by transformationmethods. The possibilities include, but are not limited to, anybiological compound which is presently produced by any organism such asproteins, nucleic acids, primary and intermediary metabolites,carbohydrate polymers, etc. The compounds may be produced by the plant,extracted upon harvest and/or processing, and used for any presentlyrecognized useful purpose such as pharmaceuticals, fragrances,industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance gamma-zein synthesis, popcorn with improved popping,quality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken gene (encoding sucrose synthase) for sweet corn.

1.7 Tuber or Seed Composition or Quality

Various traits can be advantegously expressed especially in seeds ortubers to improve composition or quality. Such traits include but arenot limited to:

-   -   Expression of metabolic enzymes for use in the food-and-feed        sector, for example of phytases and cellulases. Especially        preferred are nucleic acids such as the artificial cDNA which        encodes a microbial phytase (GenBank Acc. No.: A19451) or        functional equivalents thereof.    -   Expression of genes which bring about an accumulation of fine        chemicals such as of tocopherols, tocotrienols or carotenoids.        An example which may be mentioned is phytoene desaturase.        Preferred are nucleic acids which encode the Narcissus        pseudonarcissus photoene desaturase (GenBank Acc. No.: X78815)        or functional equivalents thereof.    -   Production of nutraceuticals such as, for example,        polyunsaturated fatty acids (for example arachidonic acid,        eicosapentaenoic acid or docosahexaenoic acid) by expression of        fatty acid elongases and/or desaturases, or production of        proteins with improved nutritional value such as, for example,        with a high content of essential amino acids (for example the        high-methionine 2S albumin gene of the brazil nut). Preferred        are nucleic acids which encode the Bertholletia excelsa        high-methionine 2S albumin (GenBank Acc. No.: AB044391), the        Physcomitrella patens Δ6-acyl-lipid desaturase (GenBank Acc.        No.: AJ222980; Girke et al. (1998) Plant J 15:39-48), the        Mortierella alpina Δ6-desaturase (Sakuradani et al. 1999 Gene        238:445-453), the Caenorhabditis elegans Δ5-desaturase        (Michaelson et al. 1998, FEBS Letters 439:215-218), the        Caenorhabditis elegans Δ5-fatty acid desaturase (des-5) (GenBank        Acc. No.: AF078796), the Mortierella alpina Δ5-desaturase        (Michaelson et al. JBC 273:19055-19059), the Caenorhabditis        elegans Δ6-elongase (Beaudoin et al. 2000, PNAS 97:6421-6426),        the Physcomitrella patens Δ6-elongase (Zank et al. 2000,        Biochemical Society Transactions 28:654-657), or functional        equivalents of these.    -   Production of high-quality proteins and enzymes for industrial        purposes (for example enzymes, such as lipases) or as        pharmaceuticals (such as, for example, antibodies, blood        clotting factors, interferons, lymphokins, colony stimulation        factor, plasminogen activators, hormones or vaccines, as        described by Hood E E, Jilka J M (1999) Curr Opin Biotechnol        10(4):382-6; Ma J K, Vine N D (1999) Curr Top Microbiol Immunol        236:275-92). For example, it has been possible to produce        recombinant avidin from chicken albumen and bacterial        b-glucuronidase (GUS) on a large scale in transgenic maize        plants (Hood et al. (1999) Adv Exp Med Biol 464:127-47. Review).    -   Obtaining an increased storability in cells which normally        comprise fewer storage proteins or storage lipids, with the        purpose of increasing the yield of these substances, for example        by expression of acetyl-CoA carboxylase. Preferred nucleic acids        are those which encode the Medicago sativa acetyl-CoA        carboxylase (ACCase) (GenBank Acc. No.: L25042), or functional        equivalents thereof.    -   Reducing levels of α-glucan L-type tuber phosphorylase (GLTP) or        .α-glucan H-type tuber phosphorylase (GHTP) enzyme activity        preferably within the potato tuber (see U.S. Pat. No.        5,998,701). The conversion of starches to sugars in potato        tubers, particularly when stored at temperatures below 7° C., is        reduced in tubers exhibiting reduced GLTP or GHTP enzyme        activity. Reducing cold-sweetening in potatoes allows for potato        storage at cooler temperatures, resulting in prolonged dormancy,        reduced incidence of disease, and increased storage life.        Reduction of GLTP or GHTP activity within the potato tuber may        be accomplished by such techniques as suppression of gene        expression using homologous antisense or double-stranded RNA,        the use of co-suppression, regulatory silencing sequences. A        potato plant having improved cold-storage characteristics,        comprising a potato plant transformed with an expression        cassette having a TPT promoter sequence operably linked to a DNA        sequence comprising at least 20 nucleotides of a gene encoding        an α-glucan phosphorylase selected from the group consisting of        α-glucan L-type tuber phosphorylase (GLTP) and α-glucan H-type        phosphorylase (GHTP).

Further examples of advantageous genes are mentioned for example inDunwell J M, Transgenic approaches to crop improvement, J Exp Bot. 2000;51 Spec No; pages 487-96.

1.8 Plant Agronomic Characteristics

Two of the factors determining where plants can be grown are the averagedaily temperature during the growing season and the length of timebetween frosts. Within the areas where it is possible to grow aparticular plant, there are varying limitations on the maximal time itis allowed to grow to maturity and be harvested. The plant to be grownin a particular area is selected for its ability to mature and dry downto harvestable moisture content within the required period of time withmaximum possible yield. Therefore, plants of varying maturities aredeveloped for different growing locations. Apart from the need to drydown sufficiently to permit harvest is the desirability of havingmaximal drying take place in the field to minimize the amount of energyrequired for additional drying post-harvest. Also the more readily thegrain can dry down, the more time there is available for growth andkernel fill. Genes that influence maturity and/or dry down can beidentified and introduced into plant lines using transformationtechniques to create new varieties adapted to different growinglocations or the same growing location but having improved yield tomoisture ratio at harvest. Expression of genes that are involved inregulation of plant development may be especially useful, e.g., theliguleless and rough sheath genes that have been identified in plants.

Genes may be introduced into plants that would improve standability andother plant growth characteristics. For example, expression of novelgenes, which confer stronger stalks, improved root systems, or preventor reduce ear droppage would be of great value to the corn farmer.Introduction and expression of genes that increase the total amount ofphotoassimilate available by, for example, increasing light distributionand/or interception would be advantageous. In addition the expression ofgenes that increase the efficiency of photosynthesis and/or the leafcanopy would further increase gains in productivity. Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilates into the grain and thus increase yield. Overexpression ofgenes within plants that are associated with “stay green” or theexpression of any gene that delays senescence would be advantageous. Forexample, a non-yellowing mutant has been identified in Festuca pratensis(Davies 1990). Expression of this gene as well as others may preventpremature breakdown of chlorophyll and thus maintain canopy function.

1.9 Nutrient Utilization

The ability to utilize available nutrients and minerals may be alimiting factor in growth of many plants. It is proposed that it wouldbe possible to alter nutrient uptake, tolerate pH extremes, mobilizationthrough the plant, storage pools, and availability for metabolicactivities by the introduction of novel genes. These modifications wouldallow a plant to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is also contemplated that expressionof a novel gene may make a nutrient source available that was previouslynot accessible, e.g., an enzyme that releases a component of nutrientvalue from a more complex molecule, perhaps a macromolecule.

1.10 Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Mariani1990). For example, a number of mutations were discovered in maize thatconfer cytoplasmic male sterility. One mutation in particular, referredto as T cytoplasm, also correlates with sensitivity to Southern cornleaf blight. A DNA sequence, designated TURF-13 (Levings 1990), wasidentified that correlates with T cytoplasm. It would be possiblethrough the introduction of TURF-13 via transformation to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility mayalso be introduced.

1.11. Non-Protein-Expressing Sequences

1.11.1 RNA-Expressing

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produceantisense RNA or double-stranded RNA that is complementary to all orpart(s) of a targeted messenger RNA(s). The antisense RNA reducesproduction of the polypeptide product of the messenger RNA. Thepolypeptide product may be any protein encoded by the plant genome. Theaforementioned genes will be referred to as antisense genes. Anantisense gene may thus be introduced into a plant by transformationmethods to produce a novel transgenic plant with reduced expression of aselected protein of interest. For example, the protein may be an enzymethat catalyzes a reaction in the plant. Reduction of the enzyme activitymay reduce or eliminate products of the reaction which include anyenzymatically synthesized compound in the plant such as fatty acids,amino acids, carbohydrates, nucleic acids and the like. Alternatively,the protein may be a storage protein, such as a zein, or a structuralprotein, the decreased expression of which may lead to changes in seedamino acid composition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

Expression of antisense-RNA or double-stranded RNA by one of theexpression cassettes of the invention is especially preferred. Alsoexpression of sense RNA can be employed for gene silencing(co-suppression). This RNA is preferably a nontranslatable RNA. Generegulation by double-stranded RNA (“double-stranded RNA interference”;dsRNAi) is well known in the arte and described for various organismincluding plants (e.g., Matzke 2000; Fire A et al 1998; WO 99/32619; WO99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO00/63364).

Genes may also be constructed or isolated, which when transcribedproduce RNA enzymes, or ribozymes, which can act as endoribonucleasesand catalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNA's can result in the reducedproduction of their encoded polypeptide products. These genes may beused to prepare novel transgenic plants, which possess them. Thetransgenic plants may possess reduced levels of polypeptides includingbut not limited to the polypeptides cited above that may be affected byantisense RNA.

It is also possible that genes may be introduced to produce noveltransgenic plants, which have reduced expression of a native geneproduct, by a mechanism of cosuppression. It has been demonstrated intobacco, tomato, and petunia (Goring 1991; Smith 1990; Napoli 1990; vander Krol 1990) that expression of the sense transcript of a native genewill reduce or eliminate expression of the native gene in a mannersimilar to that observed for antisense genes. The introduced gene mayencode all or part of the targeted native protein but its translationmay not be required for reduction of levels of that native protein.

1.11.2 Non-RNA-Expressing

For example, DNA elements including those of transposable elements suchas Ds, Ac, or Mu, may be, inserted into a gene and cause mutations.These DNA elements may be inserted in order to inactivate (or activate)a gene and thereby “tag” a particular trait. In this instance thetransposable element does not cause instability of the tagged mutation,because the utility of the element does not depend on its ability tomove in the genome. Once a desired trait is tagged, the introduced DNAsequence may be used to clone the corresponding gene, e.g., using theintroduced DNA sequence as a PCR primer together with PCR gene cloningtechniques (Shapiro, 1983; Dellaporta 1988). Once identified, the entiregene(s) for the particular trait, including control or regulatoryregions where desired may be isolated, cloned and manipulated asdesired. The utility of DNA elements introduced into an organism forpurposed of gene tagging is independent of the DNA sequence and does notdepend on any biological activity of the DNA sequence, i.e.,transcription into RNA or translation into protein. The sole function ofthe DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element, which may be introduced, is a matrixattachment region element (MAR), such as the chicken lysozyme A element(Stief 1989), which can be positioned around an expressible gene ofinterest to effect an increase in overall expression of the gene anddiminish position dependant effects upon incorporation into the plantgenome (Stief 1989; Phi-Van 1990).

Further nucleotide sequences of interest that may be contemplated foruse within the scope of the present invention in operable linkage withthe promoter sequences according to the invention are isolated nucleicacid molecules, e.g., DNA or RNA, comprising a plant nucleotide sequenceaccording to the invention comprising an open reading frame that ispreferentially expressed in a specific tissue, i.e., seed-, root, greentissue (leaf and stem), panicle-, or pollen, or is expressedconstitutively.

2. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can ‘select’ for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by ‘screening’ (e.g., the R-locus trait, the greenfluorescent protein (GFP)). Of course, many examples of suitable markergenes are known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers, which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes, whichcan be detected by their catalytic activity. Secretable proteins fallinto a number of classes, including small, diffusible proteinsdetectable, e.g., by ELISA; small active enzymes detectable inextracellular solution (e.g., alpha-amylase, beta-lactamase,phosphinothricin acetyltransferase); and proteins that are inserted ortrapped in the cell wall (e.g., proteins that include a leader sequencesuch as that found in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Steifel 1990) molecule is well characterized in terms ofmolecular biology, expression and protein structure. However, any one ofa variety of ultilane and/or glycine-rich wall proteins (Keller 1989)could be modified by the addition of an antigenic site to create ascreenable marker.

One exemplary embodiment of a secretable screenable marker concerns theuse of a maize sequence encoding the wall protein HPRG, modified toinclude a 15 residue epitope from the pro-region of murine interleukin,however, virtually any detectable epitope may be employed in suchembodiments, as selected from the extremely wide variety ofantigen-antibody combinations known to those of skill in the art. Theunique extracellular epitope can then be straightforwardly detectedusing antibody labeling in conjunction with chromogenic or fluorescentadjuncts.

Elements of the present disclosure may be exemplified in detail throughthe use of the bar and/or GUS genes, and also through the use of variousother markers. Of course, in light of this disclosure, numerous otherpossible selectable and/or screenable marker genes will be apparent tothose of skill in the art in addition to the one set forth herein below.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant.

2.1 Selectable Markers

Various selectable markers are known in the art suitable for planttransformation. Such markers may include but are not limited to:

2.1.1 Negative Selection Markers

Negative selection markers confer a resistance to a biocidal compoundsuch as a metabolic inhibitor (e.g., 2-deoxyglucose-6-phosphate, WO98145456), antibiotics (e.g., kanamycin, G 418, bleomycin or hygromycin)or herbicides (e.g., phosphinothricin or glyphosate). Transformed plantmaterial (e.g., cells, tissues or plantlets), which express markergenes, are capable of developing in the presence of concentrations of acorresponding selection compound (e.g., antibiotic or herbicide), whichsuppresses growth of an untransformed wild type tissue. Especiallypreferred negative selection markers are those, which confer resistanceto herbicides. Examples, which may be mentioned, are:

-   -   Phosphinothricin acetyltransferases (PAT; also named Bialophos®        resistance; bar; de Block 1987; Vasil 1992, 1993; Weeks 1993;        Becker 1994; Nehra 1994; Wan & Lemaux 1994; EP 0 333 033; U.S.        Pat. No. 4,975,374). Preferred are the bar gene from        Streptomyces hygroscopicus or the pat gene from Streptomyces        viridochromogenes. PAT inactivates the active ingredient in the        herbicide bialaphos, phosphinothricin (PPT). PPT inhibits        glutamine synthetase, (Murakami 1986; Twell 1989) causing rapid        accumulation of ammonia and cell death.    -   altered 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)        conferring resistance to Glyphosate®        (N-(phosphonomethyl)glycine) (Hinchee 1988; Shah 1986;        Della-Cioppa 1987). Where a mutant EPSP synthase gene is        employed, additional benefit may be realized through the        incorporation of a suitable chloroplast transit peptide, CTP        (EP-A 10 218 571).    -   Glyphosate® degrading enzymes (Glyphosate® oxidoreductase; gox),    -   Dalapon® inactivating dehalogenases (deh)    -   sulfonylurea- and/or imidazolinone-inactivating acetolactate        synthases (ahas or ALS; for example mutated ahas/ALS variants        with, for example, the S4, XI12, XA17, and/or Hra mutation        (EP-A1 154 204)    -   Bromoxynil® degrading nitrilases (bxn; Stalker 1988)    -   Kanamycin- or geneticin (G418) resistance genes (NPTII; NPT or        neo; Potrykus 1985) coding e.g., for neomycin        phosphotransferases (Fraley 1983; Nehra 1994)    -   2-Desoxyglucose-6-phosphate phosphatase (DOG^(R)1-Gene product;        WO 98/45456; EP 0 807 836) conferring resistance against        2-desoxyglucose (Randez-Gil 1995).    -   hygromycin phosphotransferase (HPT), which mediates resistance        to hygromycin (Vanden Elzen 1985).    -   altered dihydrofolate reductase (Eichholtz 1987) conferring        resistance against methotrexat (Thillet 1988);    -   mutated anthranilate synthase genes that confers resistance to        5-methyl tryptophan.

Additional negative selectable marker genes of bacterial origin thatconfer resistance to antibiotics include the aadA gene, which confersresistance to the antibiotic spectinomycin, gentamycin acetyltransferase, streptomycin phosphotransferase (SPT),aminoglycoside-3-adenyl transferase and the bleomycin resistancedeterminant (Hayford 1988; Jones 1987; Svab 1990; Hille 1986).

Especially preferred are negative selection markers that conferresistance against the toxic effects imposed by D-amino acids like e.g.,D-alanine and D-serine (WO 03/060133; Erikson 2004). Especiallypreferred as negative selection marker in this contest are the daol gene(EC: 1.4. 3.3: GenBank Acc.-No.: U60066) from the yeast Rhodotorulagracilis (Rhodosporidium toruloides) and the E. coli gene dsdA (D-serinedehydratase (D-serine deaminase) [EC: 4.3. 1.18; GenBank Acc.-No.:J01603).

Transformed plant material (e.g., cells, embryos, tissues or plantlets)which express such marker genes are capable of developing in thepresence of concentrations of a corresponding selection compound (e.g.,antibiotic or herbicide) which suppresses growth of an untransformedwild type tissue. The resulting plants can be bred and hybridized in thecustomary fashion. Two or more generations should be grown in order toensure that the genomic integration is stable and hereditary.Corresponding methods are described (Jenes 1993; Potrykus 1991).

Furthermore, reporter genes can be employed to allow visual screening,which may or may not (depending on the type of reporter gene) requiresupplementation with a substrate as a selection compound.

Various time schemes can be employed for the various negative selectionmarker genes. In case of resistance genes (e.g., against herbicides orD-amino acids) selection is preferably applied throughout callusinduction phase for about 4 weeks and beyond at least 4 weeks intoregeneration. Such a selection scheme can be applied for all selectionregimes. It is furthermore possible (although not explicitly preferred)to remain the selection also throughout the entire regeneration schemeincluding rooting.

For example, with the phosphinotricin resistance gene (bar) as theselective marker, phosphinotricin at a concentration of from about 1 to50 mg/l may be included in the medium. For example, with the dao1 geneas the selective marker, D-serine or D-alanine at a concentration offrom about 3 to 100 mg/l may be included in the medium. Typicalconcentrations for selection are 20 to 40 mg/l. For example, with themutated ahas genes as the selective marker, PURSUIT™ at a concentrationof from about 3 to 100 mg/l may be included in the medium. Typicalconcentrations for selection are 20 to 40 mg/l.

2.1.2 Positive Selection Marker

Furthermore, positive selection marker can be employed. Genes likeisopentenyltransferase from Agrobacterium tumefaciens (strain: PO22;Genbank Acc.-No.: AB025109) may—as a key enzyme of the cytokininbiosynthesis—facilitate regeneration of transformed plants (e.g., byselection on cytokinin-free medium). Corresponding selection methods aredescribed (Ebinuma 2000a,b). Additional positive selection markers,which confer a growth advantage to a transformed plant in comparisonwith a nontransformed one, are described e.g., in EP-A 0 601 092. Growthstimulation selection markers may include (but shall not be limited to)β-Glucuronidase (in combination with e.g., a cytokinin glucuronide),mannose-6-phosphate isomerase (in combination with mannose),UDP-galactose-4-epimerase (in combination with e.g., galactose), whereinmannose-6-phosphate isomerase in combination with mannose is especiallypreferred.

2.1.3 Counter-selection Marker

Counter-selection markers are especially suitable to select organismswith defined deleted sequences comprising said marker (Koprek 1999).Examples for counter-selection marker comprise thymidin kinases (TK),cytosine deaminases (Gleave 1999; Perera 1993; Stougaard 1993),cytochrom P450 proteins (Koprek 1999), haloalkan dehalogenases (Naested1999), iaaH gene products (Sundaresan 1995), cytosine deaminase codA(Schiaman & Hooykaas 1997), tms2 gene products (Fedoroff & Smith 1993),or α-naphthalene acetamide (NAM; Depicker 1988). Counter selectionmarkers may be useful in the construction of transposon tagging lines.For example, by marking an autonomous transposable element such as Ac,Master Mu, or En/Spn with a counter selection marker, one could selectfor transformants in which the autonomous element is not stablyintegrated into the genome. This would be desirable, for example, whentransient expression of the autonomous element is desired to activate intrans the transposition of a defective transposable element, such as Ds,but stable integration of the autonomous element is not desired. Thepresence of the autonomous element may not be desired in order tostabilize the defective element, i.e., prevent it from furthertransposing. However, it is proposed that if stable integration of anautonomous transposable element is desired in a plant the presence of anegative selectable marker may make it possible to eliminate theautonomous element during the breeding process.

2.2. Screenable Markers

Screenable markers that may be employed include, but are not limited to,a beta-glucuronidase (GUS) or uidA gene which encodes an enzyme forwhich various chromogenic substrates are known; an R-locus gene, whichencodes a product that regulates the production of anthocyanin pigments(red color) in plant tissues (Dellaporta 1988); a beta-lactamase gene(Sutcliffe 1978), which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylEgene (Zukowsky 1983) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta 1990); atyrosinase gene (Katz 1983) which encodes an enzyme capable of oxidizingtyrosine to DOPA and dopaquinone which in turn condenses to form theeasily detectable compound melanin; β-galactosidase gene, which encodesan enzyme for which there are chromogenic substrates; a luciferase (lux)gene (Ow 1986), which allows for bioluminescence detection; or even anaequorin gene (Prasher 1985), which may be employed in calcium-sensitivebioluminescence detection, or a green fluorescent protein gene (Niedz1995).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. A gene from the R gene complex was appliedto maize transformation, because the expression of this gene intransformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline is dominant for genes encoding the enzymatic intermediates in theanthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carriesa recessive allele at the R locus, transfomation of any cell from thatline with R will result in red pigment formation. Exemplary linesinclude Wisconsin 22 which contains the rg-Stadler allele and TR112, aK55 derivative which is r-g, b, P1. Alternatively any genotype of maizecan be utilized if the C1 and R alleles are introduced together.

It is further proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening. Where use of a screenablemarker gene such as lux or GFP is desired, benefit may be realized bycreating a gene fusion between the screenable marker gene and aselectable marker gene, for example, a GFP-NPTII gene fusion. This couldallow, for example, selection of transformed cells followed by screeningof transgenic plants or seeds.

3. Exemplary DNA Molecules

The invention provides an isolated nucleic acid molecule, e.g., DNA orRNA, comprising a plant nucleotide sequence comprising an open readingframe that is preferentially expressed in a specific plant tissue, i.e.,in seeds, roots, green tissue (leaf and stem), panicles or pollen, or isexpressed constitutively, or a promoter thereof.

These promoters include, but are not limited to, constitutive,inducible, temporally regulated, developmentally regulated,spatially-regulated, chemically regulated, stress-responsive,tissue-specific, viral and synthetic promoters. Promoter sequences areknown to be strong or weak. A strong promoter provides for a high levelof gene expression, whereas a weak promoter provides for a very lowlevel of gene expression. An inducible promoter is a promoter thatprovides for the turning on and off of gene expression in response to anexogenously added agent, or to an environmental or developmentalstimulus. A bacterial promoter such as the P_(tac) promoter can beinduced to varying levels of gene expression depending on the level ofisothiopropylgalactoside added to the transformed bacterial cells. Anisolated promoter sequence that is a strong promoter for heterologousnucleic acid is advantageous because it provides for a sufficient levelof gene expression to allow for easy detection and selection oftransformed cells and provides for a high level of gene expression whendesired.

Within a plant promoter region there are several domains that arenecessary for full function of the promoter. The first of these domainslies immediately upstream of the structural gene and forms the “corepromoter region” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence that defines thetranscription start point for the structural gene.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore, the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences upstream of the coreconstitute the remainder of the promoter. The regulatory sequencesdetermine expression level, the spatial and temporal pattern ofexpression and, for an important subset of promoters, expression underinductive conditions (regulation by external factors such as light,temperature, chemicals, hormones).

Regulated expression of the chimeric transacting viral replicationprotein can be further regulated by other genetic strategies. Forexample, Cre-mediated gene activation as described by Odell et al. 1990.Thus, a DNA fragment containing 3′ regulatory sequence bound by loxsites between the promoter and the replication protein coding sequencethat blocks the expression of a chimeric replication gene from thepromoter can be removed by Cre-mediated excision and result in theexpression of the transacting replication gene. In this case, thechimeric Cre gene, the chimeric trans-acting replication gene, or bothcan be under the control of tissue- and developmental-specific orinducible promoters. An alternate genetic strategy is the use of tRNAsuppressor gene. For example, the regulated expression of a tRNAsuppressor gene can conditionally control expression of a trans-actingreplication protein coding sequence containing an appropriatetermination codon as described by Ulmasov et al. 1997. Again, either thechimeric tRNA suppressor gene, the chimeric transacting replicationgene, or both can be under the control of tissue- anddevelopmental-specific or inducible promoters.

Frequently it is desirable to have continuous or inducible expression ofa DNA sequence throughout the cells of an organism in atissue-independent manner. For example, increased resistance of a plantt6 infection by soil- and airborne-pathogens might be accomplished bygenetic manipulation of the plant's genome to comprise a continuouspromoter operably linked to a heterologous pathogen-resistance gene suchthat pathogen-resistance proteins are continuously expressed throughoutthe plant's tissues.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within the seeds of a plant to achieve a desired phenotype.In this case, such inhibition might be accomplished with transformationof the plant to comprise a promoter operably linked to an antisensenucleotide sequence, such that seed-preferential or seed-specificexpression of the antisense sequence produces an RNA transcript thatinterferes with translation of the mRNA of the native DNA sequence.

To define a minimal promoter region, a DNA segment representing thepromoter region is removed from the 5′ region of the gene of interestand operably linked to the coding sequence of a marker (reporter) geneby recombinant DNA techniques well known to the art. The reporter geneis operably linked downstream of the promoter, so that transcriptsinitiating at the promoter proceed through the reporter gene. Reportergenes generally encode proteins, which are easily measured, including,but not limited to, chloramphenicol acetyl transferase (CAT),beta-glucuronidase (GUS), green fluorescent protein (GFP),beta-galactosidase (beta-GAL), and luciferase.

The construct containing the reporter gene under the control of thepromoter is then introduced into an appropriate cell type bytransfection techniques well known to the art. To assay for the reporterprotein, cell lysates are prepared and appropriate assays, which arewell known in the art, for the reporter protein are performed. Forexample, if CAT were the reporter gene of choice, the lysates from cellstransfected with constructs containing CAT under the control of apromoter under study are mixed with isotopically labeled chloramphenicoland acetyl-coenzyme A (acetyl-CoA). The CAT enzyme transfers the acetylgroup from acetyl-CoA to the 2- or 3-position of chloramphenicol. Thereaction is monitored by thin-layer chromatography, which separatesacetylated chloramphenicol from unreacted material. The reactionproducts are then visualized by autoradiography.

The level of enzyme activity corresponds to the amount of enzyme thatwas made, which in turn reveals the level of expression from thepromoter of interest. This level of expression can be compared to otherpromoters to determine the relative strength of the promoter understudy. In order to be sure that the level of expression is determined bythe promoter, rather than by the stability of the mRNA, the level of thereporter mRNA can be measured directly, such as by Northern blotanalysis.

Once activity is detected, mutational and/or deletional analyses may beemployed to determine the minimal region and/or sequences required toinitiate transcription. Thus, sequences can be deleted at the 5′ end ofthe promoter region and/or at the 3′ end of the promoter region, andnucleotide substitutions introduced. These constructs are thenintroduced to cells and their activity determined.

In one embodiment, the promoter may be a gamma zein promoter, an oleosinole16 promoter, a globulins promoter, an actin I promoter, an actin clpromoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter,an Ltpl promoter, an Ltp2 promoter, an oleosin ole17 promoter, anoleosin ole18 promoter, an actin 2 promoter, a pollen-specific proteinpromoter, a pollen-specific pectate lyase promoter, an anther-specificprotein promoter, an anther-specific gene RTS2 promoter, apollen-specific gene promoter, a tapeturn-specific gene promoter,tapeturn-specific gene RAB24 promoter, a anthranilate synthase alphasubunit promoter, an alpha zein promoter, an anthranilate synthase betasubunit promoter, a dihydrodipicolinate synthase promoter, a Thilpromoter, an alcohol dehydrogenase promoter, a cab binding proteinpromoter, an H3C4 promoter, a RUBISCO SS starch branching enzymepromoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, aregulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, acellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteinehydrolase promoter, a superoxide dismutase promoter, a C-kinase receptorpromoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNApromoter, a glucose-6 phosphate isomerase promoter, apyrophosphate-fructose 6-phosphatelphosphotransferase promoter, anubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDaphotosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDavacuolar ATPase subunit promoter, a metallothionein-like proteinpromoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA-and ripening-inducible-like protein promoter, a phenylalanine ammonialyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteinehydrolase promoter, an a-tubulin promoter, a cab promoter, a PEPCasepromoter, an R gene promoter, a lectin promoter, a light harvestingcomplex promoter, a heat shock protein promoter, a chalcone synthasepromoter, a zein promoter, a globulin-1 promoter, an ABA promoter, anauxin-binding protein promoter, a UDP glucose flavonoidglycosyl-transferase gene promoter, an NTI promoter, an actin promoter,an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35Spromoter, a CaMV 34S promoter, a CaMV 19S promoter, a histone promoter,a turgor-inducible promoter, a pea small subunit RuBP carboxylasepromoter, a Ti plasmid mannopine synthase promoter, Ti plasmid nopalinesynthase promoter, a petunia chalcone isomerase promoter, a bean glycinerich protein I promoter, a CaMV 35S transcript promoter, a potatopatatin promoter, or a S-E9 small subunit RuBP carboxylase promoter.

4. Transformed (Transgenic) Plants of the Invention and Methods ofPreparation

Plant species may be transformed with the DNA construct of the presentinvention by the DNA-mediated transformation of plant cell protoplastsand subsequent regeneration of the plant from the transformedprotoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis,” as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristems, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand ultilane meristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt II) can be associated with the expression cassetteto assist in breeding.

Thus, the present invention provides a transformed (transgenic) plantcell, in planta or ex planta, including a transformed plastid or otherorganelle, e.g., nucleus, mitochondria or chloroplast. The presentinvention may be used for transformation of any plant species,including, but not limited to, cells from the plant species specifiedabove in the DEFINITION section. Preferably, transgenic plants of thepresent invention are crop plants and in particular cereals (forexample, corn, alfalfa, sunflower, rice, Brassica, canola, soybean,barley, soybean, sugarbeet, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, Linum usitatissimum (linseed and fax), Camelina sativa,Brassica juncea, etc.), and even more preferably corn, rice and soybean.Other embodiments of the invention are related to cells, cell cultures,tissues, parts (such as plants organs, leaves, roots, etc.) andpropagation material (such as seeds) of such plants.

The transgenic expression cassette of the invention may not only becomprised in plants or plant cells but may advantageously also becontaining in other organisms such for example bacteria. Thus, anotherembodiment of the invention relates to transgenic cells or non-human,transgenic organisms comprising an expression cassette of the invention.Preferred are prokaryotic and eukaryotic organisms. Both microorganismand higher organisms are comprised. Preferred microorganisms arebacteria, yeast, algae, and fungi. Preferred bacteria are those of thegenus Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes,Pseudomonas, Bacillus or Cyanobacterim such as—for example—Synechocystisand other bacteria described in Brock Biology of Microorganisms EighthEdition (pages A-8, A-9, A10 and A11).

Especially preferred are microorganisms capable to infect plants and totransfer DNA into their genome, especially bacteria of the genusAgrobacterium, preferably Agrobacterium tumefaciens and rhizogenes.Preferred yeasts are Candida, Saccharomyces, Hansenula and Pichia.Preferred Fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,Fusarium, and Beauveria. Most preferred are plant organisms as definedabove.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques generally include transformation with DNA employing A.tumefaciens or A. rhizogenes as the transforming agent, liposomes, PEGprecipitation, electroporation, DNA injection, direct DNA uptake,microprojectile bombardment, particle acceleration, and the like (See,for example, EP 295959 and EP 138341) (see below). However, cells otherthan plant cells may be transformed with the expression cassettes of theinvention. The general descriptions of plant expression vectors andreporter genes, and Agrobacterium and Agrobacterium-mediated genetransfer, can be found in Gruber et al. (1993).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided for example by Maki etal., (1993); and by Phillips et al. (1988). Preferably, expressionvectors are introduced into maize or other plant tissues using a directgene transfer method such as microprojectile-mediated delivery, DNAinjection, electroporation and the like. More preferably expressionvectors are introduced into plant tissues using the microprojectilemedia delivery with the biolistic device. See, for example, Tomes et al.(1995). The vectors of the invention can not only be used for expressionof structural genes but may also be used in exon-trap cloning, orpromoter trap procedures to detect differential gene expression invarieties of tissues (Lindsey 1993; Auch & Reth 1990).

It is particularly preferred to use the binary type vectors of Ti and Riplasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti1985: Byrne 1987; Sukhapinda 1987; Lorz 1985; Potrykus, 1985; Park 1985:Hiei 1994). The use of T-DNA to transform plant cells has receivedextensive study and is amply described (EP 120516; Hoekema, 1985; Knauf,1983; and An 1985). For introduction into plants, the chimeric genes ofthe invention can be inserted into binary vectors as described in theexamples.

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 295959),techniques of electroporation (Fromm 1986) or high velocity ballisticbombardment with metal particles coated with the nucleic acid constructs(Kline 1987, and U.S. Pat. No. 4,945,050). Once transformed, the cellscan be regenerated by those skilled in the art. Of particular relevanceare the recently described methods to transform foreign genes intocommercially important crops, such as rapeseed (De Block 1989),sunflower (Everett 1987), soybean (McCabe 1988; Hinchee 1988; Chee 1989;Christou 1989; EP 301749), rice (Hiei 1994), and corn (Gordon-Kamm 1990;Fromm 1990).

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant, i.e., monocotyledonous or dicotyledonous,targeted for transformation. Suitable methods of transforming plantcells include, but are not limited to, microinjection (Crossway 1986),electroporation (Riggs 1986), Agrobacterium-mediated transformation(Hinchee 1988), direct gene transfer (Paszkowski 1984), and ballisticparticle acceleration using devices available from Agracetus, Inc.,Madison, Wis. And BioRad, Hercules, Calif. (see, for example, U.S. Pat.No. 4,945,050; and McCabe 1988). Also see, Weissinger 1988; Sanford 1987(onion); Christou 1988 (soybean); McCabe 1988 (soybean); Datta 1990(rice); Klein 1988 (maize); Klein 1988 (maize); Klein 1988 (maize);Fromm 1990 (maize); and Gordon-Kamm 1990 (maize); Svab 1990 (tobaccochloroplast); Koziel 1993 (maize); Shimamoto 1989 (rice); Christou 1991(rice); European Patent Application EP 0 332 581 (orchardgrass and otherPooideae); Vasil 1993 (wheat); Weeks 1993 (wheat).

In another embodiment, a nucleotide sequence of the present invention isdirectly transformed into the plastid genome. Plastid transformationtechnology is extensively described in U.S. Pat. Nos. 5,451,513,5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and inMcBride et al., 1994. The basic technique for chloroplast transformationinvolves introducing regions of cloned plastid DNA flanking a selectablemarker together with the gene of interest into a suitable target tissue,e.g., using biolistics or protoplast transformation (e.g., calciumchloride or PEG mediated transformation). The 1 to 1.5 kb flankingregions, termed targeting sequences, facilitate orthologousrecombination with the plastid genome and thus allow the replacement ormodification of specific regions of the plastome. Initially, pointmutations in the chloroplast 16S rRNA and rps12 genes conferringresistance to spectinomycin and/or streptomycin are utilized asselectable markers for transformation (Svab 1990; Staub 1992). Thisresulted in stable homoplasmic transformants at a frequency ofapproximately one per 100 bombardments of target leaves. The presence ofcloning sites between these markers allowed creation of aplastid-targeting vector for introduction of foreign genes (Staub 1993).Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3N-adenyltransferase (Svab 1993). Other selectablemarkers useful for plastid transformation are known in the art andencompassed within the scope of the invention. Typically, approximately15-20 cell division cycles following transformation are required toreach a homoplastidic state. Plastid expression, in which genes areinserted by orthologous recombination into all of the several thousandcopies of the circular plastid genome present in each plant cell, takesadvantage of the enormous copy number advantage over nuclear-expressedgenes to permit expression levels that can readily exceed 10% of thetotal soluble plant protein. In a preferred embodiment, a nucleotidesequence of the present invention is inserted into a plastid-targetingvector and transformed into the plastid genome of a desired plant host.Plants homoplastic for plastid genomes containing a nucleotide sequenceof the present invention are obtained, and are preferentially capable ofhigh expression of the nucleotide sequence.

Agrobacterium tumefaciens cells containing a vector comprising anexpression cassette of the present invention, wherein the vectorcomprises a Ti plasmid, are useful in methods of making transformedplants. Plant cells are infected with an Agrobacterium tumefaciens asdescribed above to produce a transformed plant cell, and then a plant isregenerated from the transformed plant cell. Numerous Agrobacteriumvector systems useful in carrying out the present invention are known.

Various Agrobacterium strains can be employed, preferably disarmedAgrobacterium tumefaciens or rhizogenes strains. In a preferredembodiment, Agrobacterium strains for use in the practice of theinvention include octopine strains, e.g., LBA4404 or agropine strains,e.g., EHA101 or EHA105. Suitable strains of A. tumefaciens for DNAtransfer are for example EHA101[pEHA101] (Hood 1986), EHA105[pEHA105](Li 1992), LBA4404[pAL4404] (Hoekema 1983), C58C1[pMP90] (Koncz & Schell1986), and C58C1[pGV2260] (Deblaere 1985). Other suitable strains areAgrobacterium tumefaciens C58, a nopaline strain. Other suitable strainsare A. tumefaciens C58C1 (Van Larebeke 1974), A136 (Watson 1975) orLBA4011 (Klapwijk 1980). In another preferred embodiment the soil-bornebacterium is a disarmed variant of Agrobacterium rhizogenes strain K599(NCPPB 2659). Preferably, these strains are comprising a disarmedplasmid variant of a Ti- or Ri-plasmid providing the functions requiredfor T-DNA transfer into plant cells (e.g., the vir genes). In apreferred embodiment, the Agrobacterium strain used to transform theplant tissue pre-cultured with the plant phenolic compound contains aL,L-succinamopine type Ti-plasmid, preferably disarmed, such as pEHA101.In another preferred embodiment, the Agrobacterium strain used totransform the plant tissue pre-cultured with the plant phenolic compoundcontains an octopine-type Ti-plasmid, preferably disarmed, such aspAL4404. Generally, when using octopine-type Ti-plasmids or helperplasmids, it is preferred that the virF gene be deleted or inactivated(Jarschow 1991).

The method of the invention can also be used in combination withparticular Agrobacterium strains, to further increase the transformationefficiency, such as Agrobacterium strains wherein the vir geneexpression and/or induction thereof is altered due to the presence ofmutant or chimeric virA or virG genes (e.g. Hansen 1994; Chen and Winans1991; Scheeren-Groot, 1994). Preferred are further combinations ofAgrobacterium tumefaciens strain LBA4404 (Hiei 1994) with super-virulentplasmids. These are preferably pTOK246-based vectors (Ishida 1996).

A binary vector or any other vector can be modified by common DNArecombination techniques, multiplied in E. coli, and introduced intoAgrobacterium by e.g., electroporation or other transformationtechniques (Mozo & Hooykaas 1991).

Agrobacterium is grown and used in a manner similar to that described inIshida (1996). The vector comprising Agrobacterium strain may, forexample, be grown for 3 days on YP medium (5 g/l yeast extract, 10 g/lpeptone, 5 g/l NaCl, 15 g/l agar, pH 6.8) supplemented with theappropriate antibiotic (e.g., 50 mg/l spectinomycin). Bacteria arecollected with a loop from the solid medium and resuspended. In apreferred embodiment of the invention, Agrobacterium cultures arestarted by use of aliquots frozen at −80° C.

The transformation of the target tissue (e.g., an immature embryo) bythe Agrobacterium may be carried out by merely contacting the targettissue with the Agrobacterium. The concentration of Agrobacterium usedfor infection and co-cultivation may need to be varied. For example, acell suspension of the Agrobacterium having a population density ofapproximately from 10⁵-10¹¹, preferably 10⁶ to 10¹⁰, more preferablyabout 10⁸ cells or cfu/ml is prepared and the target tissue is immersedin this suspension for about 3 to 10 minutes. The resulting targettissue is then cultured on a solid medium for several days together withthe Agrobacterium.

Preferably, the bacterium is employed in concentration of 10⁶ to 10¹⁰cfu/ml. In a preferred embodiment for the co-cultivation step about 1 to10 μl of a suspension of the soil-borne bacterium (e.g., Agrobacteria)in the co-cultivation medium are directly applied to each target tissueexplant and air-dried. This is saving labor and time and is reducingunintended Agrobacterium-mediated damage by excess Agrobacterium usage.

For Agrobacterium treatment, the bacteria are resuspended in a plantcompatible cocultivation medium. Supplementation of the co-culturemedium with antioxidants (e.g., silver nitrate), phenol-absorbingcompounds (like polyvinylpyrrolidone, Perl 1996) or thiol compounds(e.g., dithiothreitol, L-cysteine, Olhoft 2001) which can decreasetissue necrosis due to plant defence responses (like phenolic oxidation)may further improve the efficiency of Agrobacterium-mediatedtransformation. In another preferred embodiment, the co-cultivationmedium of comprises least one thiol compound, preferably selected fromthe group consisting of sodium thiolsulfate, dithiotrietol (DTT) andcysteine. Preferably the concentration is between about 1 mM and 10 mMof L-Cysteine, 0.1 mM to 5 mM DTT, and/or 0.1 mM to 5 mM sodiumthiolsulfate. Preferably, the medium employed during co-cultivationcomprises from about 1 μM to about 10 μM of silver nitrate and fromabout 50 mg/L to about 1,000 mg/L of L-Cystein. This results in a highlyreduced vulnerability of the target tissue againstAgrobacterium-mediated damage (such as induced necrosis) and highlyimproves overall transformation efficiency.

Various vector systems can be used in combination with Agrobacteria.Preferred are binary vector systems. Common binary vectors are based on“broad host range”-plasmids like pRK252 (Bevan 1984) or pTJS75 (Watson1985) derived from the P-type plasmid RK2. Most of these vectors arederivatives of pBIN19 (Bevan 1984). Various binary vectors are known,some of which are commercially available such as, for example, pBI101.2or pBIN19 (Clontech Laboratories, Inc. USA). Additional vectors wereimproved with regard to size and handling (e.g. pPZP; Hajdukiewicz1994). Improved vector systems are described also in WO 02/00900.

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker, which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, 1982; Bevan 1983),the bar gene which confers resistance to the herbicide phosphinothricin(White 1990, Spencer 1990), the hph gene which confers resistance to theantibiotic hygromycin (Blochlinger & Diggelmann), and the dhfr gene,which confers resistance to methotrexate (Bourouis 1983).

5. Production and Characterization of Stably Transformed Plants

Transgenic plant cells are then placed in an appropriate selectivemedium for selection of transgenic cells, which are then grown tocallus. Shoots are grown from callus. Plantlets are generated from theshoot by growing in rooting medium. The various constructs normally willbe joined to a marker for selection in plant cells. Conveniently, themarker may be resistance to a biocide (particularly an antibiotic, suchas kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide,or the like). The particular marker used will allow for selection oftransformed cells as compared to cells lacking the DNA, which has beenintroduced. Components of DNA constructs including transcriptioncassettes of this invention may be prepared from sequences, which arenative (endogenous) or foreign (exogenous) to the host. By “foreign” itis meant that the sequence is not found in the wild-type host into whichthe construct is introduced. Heterologous constructs will contain atleast one region, which is not native to the gene from which thetranscription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells andplants, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, in situ hybridizationand nucleic acid-based amplification methods such as PCR or RT-PCR orTaqMan; “biochemical” assays, such as detecting the presence of aprotein product, e.g., by immunological means (ELISAs and Western blots)or by enzymatic function; plant part assays, such as seed assays; andalso, by analyzing the phenotype of the whole regenerated plant, e.g.,for disease or pest resistance.

DNA may be isolated from cell lines or any plant parts to determine thepresence of the preselected nucleic acid segment through the use oftechniques well known to those skilled in the art. Note that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of nucleic acid elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using these technique discreet fragments of nucleic acid are amplifiedand detected by gel electrophoresis. This type of analysis permits oneto determine whether a preselected nucleic acid segment is present in astable transformant, but does not prove integration of the introducedpreselected nucleic acid segment into the host cell genome. In addition,it is not possible using PCR techniques to determine whethertransformants have exogenous genes introduced into different sites inthe, genome, i.e., whether transformants are of independent origin. Itis contemplated that using PCR techniques it would be possible to clonefragments of the host genomic DNA adjacent to an introduced preselectedDNA segment.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced preselected DNAsegments in high molecular weight DNA, i.e., confirm that the introducedpreselected, DNA segment has been integrated into the host cell genome.The technique of Southern hybridization provides information that isobtained using PCR, e.g., the presence of a preselected DNA segment, butalso demonstrates integration into the genome and characterizes eachindividual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of a preselected DNA segment.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of a preselected DNA segment to progeny. Inmost instances the characteristic Southern hybridization pattern for agiven transformant will segregate in progeny as one or more Mendeliangenes (Spencer 1992); Laursen 1994) indicating stable inheritance of thegene. The non-chimeric nature of the callus and the parentaltransformants (R₀) was suggested by germline transmission and theidentical Southern blot hybridization patterns and intensities of thetransforming DNA in callus, R₀ plants and R₁ progeny that segregated forthe transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced preselected DNA segments.In this application of PCR it is first necessary to reverse transcribeRNA into DNA, using enzymes such as reverse transcriptase, and thenthrough the use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the preselectedDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the protein products of theintroduced preselected DNA segments or evaluating the phenotypic changesbrought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to beanalyzed.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Morphological changes may include greater stature or thickerstalks. Most often changes in response of plants or plant parts toimposed treatments are evaluated under carefully controlled conditionstermed bioassays.

6. Uses of Transgenic Plants

Once an expression cassette of the invention has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.Particularly preferred plants of the invention include the agronomicallyimportant crops listed above. The genetic properties engineered into thetransgenic seeds and plants described above are passed on by sexualreproduction and can thus be maintained and propagated in progenyplants. The present invention also relates to a transgenic plant cell,tissue, organ, seed or plant part obtained from the transgenic plant.Also included within the invention are transgenic descendants of theplant as well as transgenic plant cells, tissues, organs, seeds andplant parts obtained from the descendants.

Preferably, the expression cassette in the transgenic plant is sexuallytransmitted. In one preferred embodiment, the coding sequence issexually transmitted through a complete normal sexual cycle of the R0plant to the R1 generation. Additionally preferred, the expressioncassette is expressed in the cells, tissues, seeds or plant of atransgenic plant in an amount that is different than the amount in thecells, tissues, seeds or plant of a plant, which only differs in thatthe expression cassette is absent. The transgenic plants produced hereinare thus expected to be useful for a variety of commercial and researchpurposes. Transgenic plants can be created for use in traditionalagriculture to possess traits beneficial to the grower (e.g., agronomictraits such as resistance to water deficit, pest resistance, herbicideresistance or increased yield), beneficial to the consumer of the grainharvested from the plant (e.g., improved nutritive content in human foodor animal feed; increased vitamin, amino acid, and antioxidant content;the production of antibodies (passive immunization) and nutriceuticals),or beneficial to the food processor (e.g., improved processing traits).In such uses, the plants are generally grown for the use of their grainin human or animal foods. Additionally, the use of root-specificpromoters in transgenic plants can provide beneficial traits that arelocalized in the consumable (by animals and humans) roots of plants suchas carrots, parsnips, and beets. However, other parts of the plants,including stalks, husks, vegetative parts, and the like, may also haveutility, including use as part of animal silage or for ornamentalpurposes. Often, chemical constituents (e.g., oils or starches) of maizeand other crops are extracted for foods or industrial use and transgenicplants may be created which have enhanced or modified levels of suchcomponents.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules. The transgenic plants may also be used incommercial breeding programs, or may be crossed or bred to plants ofrelated crop species. Improvements encoded by the expression cassettemay be transferred, e.g., from maize cells to cells of other species,e.g., by protoplast fusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences which can be used to identify proprietary linesor varieties.

Thus, the transgenic plants and seeds according to the invention can beused in plant breeding, which aims at the development of plants withimproved properties conferred by the expression cassette, such astolerance of drought, disease, or other stresses. The various breedingsteps are characterized by well-defined human intervention such asselecting the lines to be crossed, directing pollination of the parentallines, or selecting appropriate descendant plants. Depending on thedesired properties different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, multilane breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical orbiochemical means. Cross-pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines, which for exampleincrease the effectiveness of conventional methods such as herbicide orpesticide treatment or allow dispensing with said methods due to theirmodified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained which, due to their optimized genetic“equipment”, yield harvested product of better quality than products,which were not able to tolerate comparable adverse developmentalconditions.

EXAMPLES

Materials and General Methods

Unless indicated otherwise, chemicals and reagents in the Examples wereobtained from Sigma Chemical Company (St. Louis, Mo.), restrictionendonucleases were from New England Biolabs (Beverly, Mass.) or Roche(Indianapolis, Ind.), oligonucleotides were synthesized by MWG BiotechInc. (High Point, N.C.), and other modifying enzymes or kits regardingbiochemicals and molecular biological assays were from Clontech (PaloAlto, Calif.), Pharmacia Biotech (Piscataway, N.J.), Promega Corporation(Madison, Wis.), or Stratagene (La Jolla, Calif.). Materials for cellculture media were obtained from Gibco/BRL (Gaithersburg, Md.) or DIFCO(Detroit, Mich.). The cloning steps carried out for the purposes of thepresent invention, such as, for example, restriction cleavages, agarosegel electrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linking DNA fragments,transformation of E. coli cells, growing bacteria, multiplying phagesand sequence analysis of recombinant DNA, are carried out as describedby Sambrook (1989). The sequencing of recombinant DNA molecules iscarried out using ABI laser fluorescence DNA sequencer following themethod of Sanger (Sanger 1977).

Example 1 Generation of Transgenic Plants

1.1 Generation of Transgenic Arabidopsis thaliana Plants

For generating transgenic Arabidopsis plants Agrobacterium tumefaciens(strain C58C1[pMP90]) is transformed with the various promoter::GUSvector constructs (see below). Resulting Agrobacterium strains aresubsequently employed to obtain transgenic plants. For this purpose aisolated transformed Agrobacterium colony is incubated in 4 ml culture(Medium: YEB medium with 50 μg/ml Kanamycin and 25 μg/ml Rifampicin)over night at 28° C. With this culture a 400 ml culture of the samemedium is inoculated and incubated over night (28° C., 220 rpm). Thebacteria a precipitated by centrifugation (GSA-Rotor, 8.000 U/min, 20min) and the pellet is resuspended in infiltration medium (1/2MS-Medium; 0.5 g/l MES, pH 5.8; 50 g/l sucrose). The suspension isplaced in a plant box (Duchefa) and 100 ml SILVET L-77 (Osi Special-tiesInc., Cat. P030196) are added to a final concentration of 0.02%. Theplant box with 8 to 12 Plants is placed into an exsiccator for 10 to 15min. under vacuum with subsequent, spontaneous ventilation (expansion).This process is repeated 2-3 times. Thereafter all plants aretransferred into pods with wet-soil and grown under long daytimeconditions (16 h light; day temperature 22-24° C., night temperature 19°C.; 65% rel. humidity). Seeds are harvested after 6 weeks.

1.2 Generation of Transgenic Linseed

Transgenic linseed plants can be generated for example by the method ofBell et al., 1999, In Vitro Cell. Dev. Biol.-Plant. 35(6):456-465 bymeans of particle bombardment. Agrobacteria-mediated transformations canbe generated for example by the method of Mlynarova et al. (1994), PlantCell Report 13: 282-285.

Example 2 Growth Conditions for Plants for Tissue-specific ExpressionAnalysis

To obtain 4 and 7 days old seedlings, about 400 seeds (Arabidopsisthaliana ecotype Columbia) are sterilized with a 80% (v/v) ethanol:watersolution for 2 minutes, treated with a sodium hypochlorite solution(0.5% v/v) for 5 minutes, washed three times with distillated water andincubated at 4° C. for 4 days to ensure a standardized germination.Subsequently, seeds are incubated on Petri dishes with MS medium (SigmaM5519) supplemented with 1% sucrose, 0.5 g/l MES (Sigma M8652), 0.8%Difco-BactoAgar (Difco 0140-01), adjusted to pH 5.7. The seedlings aregrown under 16 h light/8 h dark cyklus (Philips 58W/33 white light) at22° C. and harvested after 4 or 7 days, respectively.

To obtain root tissue, 100 seeds are sterilized as described above,incubated at 4° C. for 4 days, and transferred into 250 ml flasks withMS medium (Sigma M5519) supplemented with additional 3% sucrose and 0.5g/l MES (Sigma M8652), adjusted to pH 5.7 for further growing. Theseedlings are grown at a 16 h light/8 h dark cycle (Philips 58W/33 whitelight) at 22° C. and 120 rpm and harvested after 3 weeks. For all otherplant organs employed, seeds are sown on standard soil (Type VM,Manna-Italia, Via S. Giacomo 42, 39050 San Giacomo/Laives, Bolzano,Italien), incubated for 4 days at 4° C. to ensure uniform germination,and subsequently grown under a 16 h light/8 darkness regime (OSRAMLumi-lux Daylight 36W/12) at 22° C. Young rosette leaves are harvestedat the 8-leaf stage (after about 3 weeks), mature rosette leaves areharvested after 8 weeks briefly before stem formation. Apices ofout-shooting stems are harvested briefly after out-shooting. Stem, stemleaves, and flower buds are harvested in development stage 12 (Bowmann J(ed.), Arabidopsis, Atlas of Morphology, Springer New York, 1995) priorto stamen development. Open flowers are harvested in development stage14 immediately after stamen development. Wilting flowers are harvestedin stage 15 to 16. Green and yellow shoots used for the analysis have alength of 10 to 13 mm.

Example 3 Demonstration of Expression Profile

To demonstrate and analyze the transcription regulating properties of apromoter of the useful to operably link the promoter or its fragments toa reporter gene, which can be employed to monitor its expression bothqualitatively and quantitatively. Preferably bacterial β-glucuronidaseis used (Jefferson 1987). β-glucuronidase activity can be monitored inplanta with chromogenic substrates such as5-bromo-4-Chloro-3-indolyl-β-D-glucuronic acid during correspondingactivity assays (Jefferson 1987). For determination of promoter activityand tissue specificity plant tissue is dissected, embedded, stained andanalyzed as described (e.g., Bäumlein 1991).

For quantitative β-glucuronidase activity analysis MUG(methylumbelliferyl glucuronide) is used as a substrate, which isconverted into MU (methylumbelliferone) and glucuronic acid. Underalkaline conditions this conversion can be quantitatively monitoredfluorometrically (excitation at 365 nm, measurement at 455 nm;SpectroFluorimeter Thermo Life Sciences Fluoroscan) as described (Bustos1989).

Example 4 Cloning of the Promoter Fragments

To isolate the promoter fragments described by SEQ ID NO: 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 28,31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 50, 51, 52,53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 74,75, 76, 77, 80, 81, 84, 85, 86, 87, 148, 149, 150, 151, 152, 153, 154,155, 158, 159, 160, 161, 162, 163, 164, 165, 168, 169, 170, 171, 172,173, 174, 175, 178, 179, 180, 181, 182, 183, 184, and 185, genomic DNAis isolated from Arabidopsis thaliana (ecotype Columbia) as described(Galbiati 2000). The isolated genomic DNA is employed as matrix DNA fora polymerase chain reaction (PCR) mediated amplification using theoligonucleotide primers and protocols indicated below (Table 3).

TABLE 3 PCR oligonucleotide primers for amplification of the varioustranscription regulating nucleotide sequences and restriction enzymesfor modifying the resulting PCR products Forward Reverse Restriction SEQID Promoter Primer Primer enzymes SEQ ID NO: 1 pSUK222L SUK222forSUK222Lrev BamHI/NcoI SEQ ID NO: 90 SEQ ID NO: 91 SEQ ID NO: 2pSUK222LGB SUK222for SUK222Lrev BamHI/NcoI SEQ ID NO: 90 SEQ ID NO: 91SEQ ID NO: 3 pSUK222S SUK222for SUK222Srev BamHI/NcoI SEQ ID NO: 90 SEQID NO: 92 SEQ ID NO: 4 pSUK222SGB SUK222for SUK222Srev BamHI/NcoI SEQ IDNO: 90 SEQ ID NO: 92 SEQ ID NO: 5 pSUK224L SUK224for SUK224LrevBamHI/NcoI SEQ ID NO: 93 SEQ ID NO: 94 SEQ ID NO: 6 pSUK224LGB SUK224forSUK224Lrev BamHI/NcoI SEQ ID NO: 93 SEQ ID NO: 94 SEQ ID NO: 7 pSUK224SSUK224for SUK224Srev BamHI/NcoI SEQ ID NO: 93 SEQ ID NO: 95 SEQ ID NO: 8pSUK224SGB SUK224for SUK224Srev BamHI/NcoI SEQ ID NO: 93 SEQ ID NO: 95SEQ ID NO: 9 pSUK226L SUK226for SUK226Lrev BamHI/NcoI SEQ ID NO: 96 SEQID NO: 97 SEQ ID NO: 10 pSUK226LGB SUK226for SUK226Lrev BamHI/NcoI SEQID NO: 96 SEQ ID NO: 97 SEQ ID NO: 11 pSUK226S SUK226for SUK226SrevBamHI/NcoI SEQ ID NO: 96 SEQ ID NO: 98 SEQ ID NO: 12 pSUK226SGBSUK226for SUK226Srev BamHI/NcoI SEQ ID NO: 96 SEQ ID NO: 98 SEQ ID NO:15 pSUK322L SUK322for SUK322Lrev XhoI/BamHI SEQ ID NO: 99 SEQ ID NO: 100SEQ ID NO: 16 pSUK322LGB SUK322for SUK322Lrev XhoI/BamHI SEQ ID NO: 99SEQ ID NO: 100 SEQ ID NO: 17 pSUK322S SUK322for SUK322Srev XhoI/HindIIISEQ ID NO: 99 SEQ ID NO: 101 SEQ ID NO: 18 pSUK322SGB SUK322forSUK322Srev XhoI/HindIII SEQ ID NO: 99 SEQ ID NO: 101 SEQ ID NO: 19pSUK324LGB SUK324for SUK324Lrev EcoRI/EcoRI SEQ ID NO: 102 SEQ ID NO:103 SEQ ID NO: 20 pSUK324SGB SUK324for SUK324Srev EcoRI/HindIII SEQ IDNO: 102 SEQ ID NO: 104 SEQ ID NO: 23 pSUK250L SUK250for SUK250LrevBamHI/NcoI SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 24 pSUK250LGBSUK250for SUK250Lrev BamHI/NcoI SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO:25 pSUK250S SUK250for SUK250Srev BamHI/NcoI SEQ ID NO: 105 SEQ ID NO:107 SEQ ID NO: 26 pSUK250SGB SUK250for SUK250Srev BamHI/NcoI SEQ ID NO:105 SEQ ID NO: 107 SEQ ID NO: 27 pSUK252LGB SUK252for SUK252LrevBamHI/NcoI SEQ ID NO: 108 SEQ ID NO: 109 SEQ ID NO: 28 pSUK252SGBSUK252for SUK252Srev BamHI/NcoI SEQ ID NO: 108 SEQ ID NO: 110 SEQ ID NO:31 pSUK19S SUK19Sfor SUK19Srev HindIII/XbaI SEQ ID NO: 111 SEQ ID NO:112 SEQ ID NO: 32 pSUK19SGB SUK19Sfor SUK19Srev HindIII/XbaI SEQ ID NO:111 SEQ ID NO: 112 SEQ ID NO: 33 pSUK19LGB SUK19Lfor SUK19LrevBamHI/NcoI SEQ ID NO: 113 SEQ ID NO: 114 SEQ ID NO: 36 pSUK28L SUK28forSUK28Lrev SalI/SmaI SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 37pSUK28LGB SUK28for SUK28Lrev SalI/SmaI SEQ ID NO: 115 SEQ ID NO: 116 SEQID NO: 38 pSUK28S SUK28for SUK28Srev SalI/XhoI SEQ ID NO: 115 SEQ ID NO:117 SEQ ID NO: 39 pSUK28SGB SUK28for SUK28Srev SalI/XhoI SEQ ID NO: 115SEQ ID NO: 117 SEQ ID NO: 40 pSUK29L SUK29for SUK29Lrev SalI/SmaI SEQ IDNO: 118 SEQ ID NO: 119 SEQ ID NO: 41 pSUK29LGB SUK29for SUK29LrevSalI/SmaI SEQ ID NO: 118 SEQ ID NO: 119 SEQ ID NO: 42 pSUK29S SUK29forSUK29Srev SalI/XhoI SEQ ID NO: 118 SEQ ID NO: 120 SEQ ID NO: 43pSUK29SGB SUK29for SUK29Srev SalI/XhoI SEQ ID NO: 118 SEQ ID NO: 120 SEQID NO: 44 pSUK30L SUK30for SUK30Lrev BamHI/NcoI SEQ ID NO: 121 SEQ IDNO: 122 SEQ ID NO: 45 pSUK30LGB SUK30for SUK30Lrev BamHI/NcoI SEQ ID NO:121 SEQ ID NO: 122 SEQ ID NO: 46 pSUK30S SUK30for SUK30Srev BamHI/XhoISEQ ID NO: 121 SEQ ID NO: 123 SEQ ID NO: 47 pSUK30SGB SUK30for SUK30SrevBamHI/XhoI SEQ ID NO: 121 SEQ ID NO: 123 SEQ ID NO: 50 pSUK298LSUK298for SUK298Lrev BamHI/NcoI SEQ ID NO: 124 SEQ ID NO: 125 SEQ ID NO:51 pSUK298LGB SUK298for SUK298Lrev BamHI/NcoI SEQ ID NO: 124 SEQ ID NO:125 SEQ ID NO: 52 pSUK298S SUK298for SUK298Srev BamHI/NcoI SEQ ID NO:124 SEQ ID NO: 126 SEQ ID NO: 53 pSUK298SGB SUK298for SUK298SrevBamHI/NcoI SEQ ID NO: 124 SEQ ID NO: 126 SEQ ID NO: 54 pSUK300LSUK300for SUK300Lrev BamHI/NcoI SEQ ID NO: 127 SEQ ID NO: 128 SEQ ID NO:55 pSUK300LGB SUK300for SUK300Lrev BamHI/NcoI SEQ ID NO: 127 SEQ ID NO:128 SEQ ID NO: 56 pSUK300S SUK300for SUK300Srev BamHI/NcoI SEQ ID NO:127 SEQ ID NO: 129 SEQ ID NO: 57 pSUK300SGB SUK300for SUK300SrevBamHI/NcoI SEQ ID NO: 127 SEQ ID NO: 129 SEQ ID NO: 60 pSUK292LSUK292for SUK292Lrev BamHI/NcoI SEQ ID NO: 130 SEQ ID NO: 131 SEQ ID NO:61 pSUK292LGB SUK292for SUK292Lrev BamHI/NcoI SEQ ID NO: 130 SEQ ID NO:131 SEQ ID NO: 62 pSUK292S SUK292for SUK292Srev BamHI/NcoI SEQ ID NO:130 SEQ ID NO: 132 SEQ ID NO: 63 pSUK292SGB SUK292for SUK292SrevBamHI/NcoI SEQ ID NO: 130 SEQ ID NO: 132 SEQ ID NO: 64 pSUK294LSUK294for SUK294Lrev BamHI/NcoI SEQ ID NO: 133 SEQ ID NO: 134 SEQ ID NO:65 pSUK294LGB SUK294for SUK294Lrev BamHI/NcoI SEQ ID NO: 133 SEQ ID NO:134 SEQ ID NO: 66 pSUK294S SUK294for SUK294Srev BamHI/NcoI SEQ ID NO:133 SEQ ID NO: 135 SEQ ID NO: 67 pSUK294SGB SUK294for SUK294SrevBamHI/NcoI SEQ ID NO: 133 SEQ ID NO: 135 SEQ ID NO: 68 pSUK296LSUK296for SUK296Lrev BamHI/NcoI SEQ ID NO: 136 SEQ ID NO: 137 SEQ ID NO:69 pSUK296LGB SUK296for SUK296Lrev BamHI/NcoI SEQ ID NO: 136 SEQ ID NO:137 SEQ ID NO: 70 pSUK296S SUK296for SUK296Srev BamHI/NcoI SEQ ID NO:136 SEQ ID NO: 138 SEQ ID NO: 71 pSUK296SGB SUK296for SUK296SrevBamHI/NcoI SEQ ID NO: 136 SEQ ID NO: 138 SEQ ID NO: 74 pSUK262LSUK262for SUK262Lrev BamHI/NcoI SEQ ID NO: 139 SEQ ID NO: 140 SEQ ID NO:75 pSUK262LGB SUK262for SUK262Lrev BamHI/NcoI SEQ ID NO: 139 SEQ ID NO:140 SEQ ID NO: 76 pSUK262S SUK262for SUK262Srev BamHI/NcoI SEQ ID NO:139 SEQ ID NO: 141 SEQ ID NO: 77 pSUK262SGB SUK262for SUK262SrevBamHI/NcoI SEQ ID NO: 139 SEQ ID NO: 141 SEQ ID NO: 80 pSUK164 SUK164forSUK164rev BamHI/NcoI SEQ ID NO: 142 SEQ ID NO: 143 SEQ ID NO: 81pSUK164GB SUK164for SUK164rev BamHI/NcoI SEQ ID NO: 142 SEQ ID NO: 143SEQ ID NO: 84 pSUK352L SUK352for SUK352Lrev BamHI/NcoI SEQ ID NO: 144SEQ ID NO: 145 SEQ ID NO: 85 pSUK352LGB SUK352for SUK352Lrev BamHI/NcoISEQ ID NO: 144 SEQ ID NO: 145 SEQ ID NO: 86 pSUK352S SUK352forSUK352Srev BamHI/NcoI SEQ ID NO: 144 SEQ ID NO: 146 SEQ ID NO: 87pSUK352SGB SUK352for SUK352Srev BamHI/NcoI SEQ ID NO: 144 SEQ ID NO: 146SEQ ID NO: 148 pSUK40L SUK40for SUK40Lrev BamHI/NcoI SEQ ID NO: 188 SEQID NO: 189 SEQ ID NO: 149 pSUK40LGB SUK40for SUK40Lrev BamHI/NcoI SEQ IDNO: 188 SEQ ID NO: 189 SEQ ID NO: 150 pSUK40S SUK40for SUK40SrevBamHI/NcoI SEQ ID NO: 188 SEQ ID NO: 190 SEQ ID NO: 151 pSUK40SGBSUK40for SUK40Srev BamHI/NcoI SEQ ID NO: 188 SEQ ID NO: 190 SEQ ID NO:152 pSUK42L SUK42for SUK42Lrev BamHI/NcoI SEQ ID NO: 191 SEQ ID NO: 192SEQ ID NO: 153 pSUK42LGB SUK42for SUK42Lrev BamHI/NcoI SEQ ID NO: 191SEQ ID NO: 192 SEQ ID NO: 154 pSUK42S SUK42for SUK42Srev BamHI/NcoI SEQID NO: 191 SEQ ID NO: 193 SEQ ID NO: 155 pSUK42SGB SUK42for SUK42SrevBamHI/NcoI SEQ ID NO: 191 SEQ ID NO: 193 SEQ ID NO: 158 pSUK48L SUK48forSUK48Lrev BamHI/NcoI SEQ ID NO: 194 SEQ ID NO: 195 SEQ ID NO: 159pSUK48LGB SUK48for SUK48Lrev BamHI/NcoI SEQ ID NO: 194 SEQ ID NO: 195SEQ ID NO: 160 pSUK48S SUK48for SUK48Srev BamHI/NcoI SEQ ID NO: 194 SEQID NO: 196 SEQ ID NO: 161 pSUK48SGB SUK48for SUK48Srev BamHI/NcoI SEQ IDNO: 194 SEQ ID NO: 196 SEQ ID NO: 162 pSUK50L SUK50for SUK50LrevBamHI/NcoI SEQ ID NO: 197 SEQ ID NO: 198 SEQ ID NO: 163 pSUK50LGBSUK50for SUK50Lrev BamHI/NcoI SEQ ID NO: 197 SEQ ID NO: 198 SEQ ID NO:164 pSUK50S SUK50for SUK50Srev BamHI/NcoI SEQ ID NO: 197 SEQ ID NO: 199SEQ ID NO: 165 pSUK50SGB SUK50for SUK50Srev BamHI/NcoI SEQ ID NO: 197SEQ ID NO: 199 SEQ ID NO: 168 pSUK96L SUK96for SUK96Lrev XhoI/NcoI SEQID NO: 200 SEQ ID NO: 201 SEQ ID NO: 169 pSUK96LGB SUK96for SUK96LrevXhoI/NcoI SEQ ID NO: 200 SEQ ID NO: 201 SEQ ID NO: 170 pSUK96S SUK96forSUK96Srev XhoI/NcoI SEQ ID NO: 200 SEQ ID NO: 202 SEQ ID NO: 171pSUK96SSGB SUK96for SUK96Srev XhoI/NcoI SEQ ID NO: 200 SEQ ID NO: 202SEQ ID NO: 172 pSUK98L SUK98for SUK98Lrev XhoI/NcoI SEQ ID NO: 203 SEQID NO: 204 SEQ ID NO: 173 pSUK98LGB SUK98for SUK98rev XhoI/NcoI SEQ IDNO: 203 SEQ ID NO: 204 SEQ ID NO: 174 pSUK98S SUK98for SUK98SrevXhoI/NcoI SEQ ID NO: 203 SEQ ID NO: 205 SEQ ID NO: 175 pSUK98SGBSUK98for SUK98Srev XhoI/NcoI SEQ ID NO: 203 SEQ ID NO: 205 SEQ ID NO:178 pSUK152L SUK152for SUK152Lrev BamHI/NcoI SEQ ID NO: 206 SEQ ID NO:207 SEQ ID NO: 179 pSUK152LGB SUK152for SUK152Lrev BamHI/NcoI SEQ ID NO:206 SEQ ID NO: 207 SEQ ID NO: 180 pSUK152S SUK152for SUK152SrevBamHI/NcoI SEQ ID NO: 206 SEQ ID NO: 208 SEQ ID NO: 181 pSUK152SGBSUK152for SUK152Srev BamHI/NcoI SEQ ID NO: 206 SEQ ID NO: 208 SEQ ID NO:182 pSUK154L SUK154for SUK154Lrev BamHI/NcoI SEQ ID NO: 209 SEQ ID NO:210 SEQ ID NO: 183 pSUK154LGB SUK154for SUK154Lrev BamHI/NcoI SEQ ID NO:209 SEQ ID NO: 210 SEQ ID NO: 184 pSUK154S SUK154for SUK154SrevBamHI/NcoI SEQ ID NO: 209 SEQ ID NO: 211 SEQ ID NO: 185 pSUK154SGBSUK154for SUK154Srev BamHI/NcoI SEQ ID NO: 209 SEQ ID NO: 211

Amplification is carried out as follows:

100 ng genomic DNA

1×PCR buffer

2.5 mM MgCl₂,

200 pM each of dATP, dCTP, dGTP und dTTP

10 pmol of each oligonucleotide primers

2.5 Units Pfu DNA Polymerase (Stratagene)

in a final volume of 50 μl

The following temperature program is employed for the variousamplifications (BIORAD Thermocycler).

1. 95° C. for 5 min

2. 54° C. for 1 min, followed by 72° C. for 5 min and 95° C. for 30 sec.Repeated 25 times.

3. 54° C. for 1 min, followed by 72° C. for 10 min.

4. Storage at 4° C.

The resulting PCR-products are digested with the restrictionendonucleases specified in the Table above (Table 3) and cloned into thevector pSUN0301 (SEQ ID NO: 147) (pre-digested with the same enzymes)upstream and in operable linkage to the glucuronidase (GUS) gene.Following stable transformation of each of these constructs intoArabidopsis thaliana tissue specificity and expression profile wasanalyzed by a histochemical and quantitative GUS-assay, respectively.

Example 5 Expression Profile of the Various Promoter::GUS Constructs inStably Transformed A. thaliana Plants

5.1 pSUK222L, pSUK222LGB, pSUK222S, pSUK222SGB, pSUK224L, pSUK224LGB,pSUK224S, pSUK224SGB, pSUK226L, pSUK226LGB, pSUK226S, pSUK226SGB

All promoter fragments conferred seed-preferred expression to thereporter gene construct tested. GUS expression was not limited toembryos but covered also seed coat and endosperm tissue. GUS expressionwas detected early in seed development and continued into maturingsiliques. Promoter activity was also observed in various organs andtissues of adult plants at varying strength, e.g. in hydathodes ofrosette and caudal leaves, vessels of stalks and rosette leaves, andalso in sepals, petals and pollen. GUS expression in seedlings wasdetected in vasculature of rosette leaves as well as in the centralcylinder of roots and also in root tips. These side activities wereleast apparent while expression in seeds was strongest for the longestpromoter fragment analyzed.

5.2 pSUK322L, pSUK322LGB, pSUK322S, pSUK322SGB, pSUK324LGB, pSUK324SGB

These promoter fragments conferred seed-preferred GUS expression. GUSactivity in seeds including expression in embryos became apparent earlyduring silique development and continued well into later stages. GUSexpression was also detected at strongly varying degrees in rosetteleaves and lateral roots of seedlings, as well as rosette leaves,stalks, siliques and anthers (mainly pollen) of adult plants analyzed.

5.3 pSUK250L, pSUK250LGB, pSUK250S, pSUK250SGB, pSUK252LGB, pSUK252SGB

Seed-preferred GUS expression conferred by the promoters analyzedcommenced early in seed development and continued well into maturesiliques. The promoters were found to be active only partially inembryos but strongly in seed coat and endosperm. In seedlings, sideactivities were mainly detected in petioles and leaf tips as well asroot tips while GUS expression in adult plants was highly specific forseeds.

5.4 pSUK19S, pSUK19SGB, pSUK19LGB

Rather strong and seed-specific GUS expression was detected late duringseed development in maturing siliques. The promoters were predominantlyactive in embryos but side activities were also observed at cotyledonsof seedlings as well as guard cells of stalks and siliques and (rarely)in midveins of rosette leaves and anthers.

5.5 pSUK28L, pSUK28LGB, pSUK28S, pSUK28SGB, pSUK29L, pSUK29LGB, pSUK29S,pSUK29SGB, pSUK30L, pSUK30LGB, pSUK30S, pSUK30SGB

Embryos as well as seed coat and endosperm in late developing to matureseeds revealed GUS expression driven by all promoters analyzed. WhilepSUK28L and pSUK30L were also characterized by promoter side activity invascular tissue of stalks and leaves of adult plants as well as siliqueand flower bottoms and mainly vascular tissue of seedlings, pSUK29L washighly seed-specific in its expression pattern observed.

5.6 pSUK298L, pSUK298LGB, pSUK298S, pSUK298SGB, pSUK300L, pSUK300LGB,pSUK300S, pSUK300SGB

These promoter fragments drive expression in entire seeds, i.e. embryosand seed coats as well as endosperm are affected. However, promoteractivity is confined to an intermediate developmental phase of theseeds, i.e. neither young nor completely mature seeds reveal GUSexpression. Side activities occur in patchy styles in hypocotyl,petioles and vascular leaf tissue of seedlings as well as in stalks,siliques, rosette leaves and floral organs of full-grown plants. Theseside activities are less pronounced for the shorter promoter fragmentpSUK298L compared with pSUK300L.

5.7 pSUK292L, pSUK292LGB, pSUK292S, pSUK292SGB, pSUK294L, pSUK294LGB,pSUK294S, pSUK294SGB, pSUK296L, pSUK296LGB, pSUK296S, pSUK296SGB

Reporter gene expression was driven by all promoter fragments inembryos, endosperm and seed coat. GUS activity commenced rather early inseed development and continued into mature seeds. However, multiple sideactivities were observed, in particular for the longer promoterfragments analyzed. The most prominent side activity was obvious inguard cells, but other organs of seedlings and adult plants transformedwith reporter gene constructs harboring the longer promoter variantsalso clearly showed GUS expression (e.g. in styles, trichomes).

5.8 pSUK262L, pSUK262LGB, pSUK262S, pSUK262SGB

These weak to medium-strong promoters are active in maturing seeds whileexpression was not detected in very young and completely mature seeds.Reporter gene expression was observed in seed coat and endosperm butcould not be proven for the embryo. Side activity is marginal comparedto other promoters disclosed: Expression apart from the seeds becameonly apparent for sepals, midveins and hydathodes of adult plants andseedlings analyzed.

5.9 pSUK164, pSUK164GB

Promoter activity was detected in embryo, endosperm and seed coat ofseeds starting early in development and continuing into the maturingphase. Side activities were observed in cotyledons and first rosetteleaves of seedlings as well as in vascular tissue of rosette leaves ofadult plants and in pollen.

5.10 pSUK352L, pSUK352LGB, pSUK352S, pSUK352SGB

Promoter activity commences early in seed development and ceases beforefull maturation. Expression is specific for embryos, i.e. no reportergene activity was detected in seeds coat or endosperm. Weak to mediumside activity was also observed in cotyledons and leaves of seedlings.

5.11 pSUK40L, pSUK40LGB, pSUK40S, pSUK40SGB, pSUK42L, pSUK42LGB,pSUK42S, pSUK42SGB

These promoters confer seed-preferential expression when tested inArabidopsis thaliana. Expression is strongest in young seeds and coversthe developing embryo as well as the seed coat. Side activities areobserved in root tips, rarely in vascular tissue of pods and stalks andalso in anthers. Longer promoter fragments confer stronger expression inseeds but are also accompanied by more pronounced side activities.

5.12 pSUK48L, pSUK48LGB, pSUK48S, pSUK48SGB, pSUK50L, pSUK50LGB,pSUK50S, pSUK50SGB

These promoters drive seed-preferential expression starting at ratherearly but maintained also in later stages of seed development ofArabidopsis thaliana. Endosperm and seed coat clearly revealed reportergene activity while it could not been shown that these promoters arealso active in embryos. Whereas the shorter promoter fragments (pSUK48derivatives) are almost void of side activity apart from some expressionobserved in vascular tissue of stalks and rarely observed expression inroot tips, pSUK50 derivatives are also characterized by varying degreesof reporter gene activity in various organs and tissues analyzed.

5.13 pSUK96L, pSUK96LGB, pSUK96S, pSUK96SGB, pSUK98L, pSUK98LGB,pSUK98S, pSUK98SGB

These promoters confer seed-preferential expression in Arabidopsisthaliana. Expression in seed coat is stronger than in any other seedtissue. Side activity of the promoters was observed mainly in vasculartissue of stalks, ovaries and siliques but also in roots (particularlyroot tips). Those side activity but also the expression detected inseeds was stronger with longer promoter fragments (pSUK98 derivatives)where expression of the marker gene was also visible in vascular tissueof flowers and leaves of a number of plants analyzed.

5.14 pSUK152L, pSUK152LGB, pSUK152S, pSU K152SGB, pSU K154L, pSUK154LGB,pSUK154S, pSUK154SGB

These promoters drive strong seed-preferential expression of the markergene analyzed in Arabidopsis thaliana. However, expression is weaker ornot detectable in very young and fully mature seeds. The promoters areactive in endosperm and embryo but not in the seed coat. The strengthand variation of side activity does not depend on the length of thepromoter analyzed. Marker gene expression was (besides seeds) observedin roots, apical meristems and petioles of seedlings, axillary meristemsand vascular tissue of stalks of adult plants as well as in variousflower organs.

Example 6 Expression Profile of the Various Promoter::GUS Constructs inStably Transformed Linseed Plants

6.1 pSUK40L

pSUK40L is a seed-specific promoter in linseed. No GUS expression wasdetected in early and young embryos. Middle strong to strong GUSexpression was found in middle, late and mature embryos.

6.2 pSUK48L, and pSUK50L

pSUK48L is a seed-specific promoter in linseed. No GUS expression wasdetected in early and young embryos. Middle strong to strong GUSexpression was found in middle, late and mature embryos.

pSUK50L is a seed-specific promoter in linseed. No GUS expression wasdetected in early and young embryos. Middle strong GUS expression wasfound mid-phase during embryo development. Middle strong to strong GUSexpression was found in late and mature embryos.

6.3 pSUK96L

pSUK96L is a seed-specific promoter in linseed. No GUS expression wasdetected in early and young embryos. Weak to medium-strong expressionwas found in medium, late and mature embryos.

6.4 pSUK152L

pSUK152L is a seed-specific promoter in linseed. No to very weak GUSexpression was detected in early and young embryos. Middle strong tostrong GUS expression was found in middle, late and mature embryos.Often medium strong to strong GUS expression was also found in the seedcapsula and in the flower.

Example 7 Vector Construction for Overexpression and Gene “Knockout”Experiments

7.1 Overexpression

Vectors used for expression of full-length “candidate genes” of interestin plants (overexpression) are designed to overexpress the protein ofinterest and are of two general types, biolistic and binary, dependingon the plant transformation method to be used.

For biolistic transformation (biolistic vectors), the requirements areas follows:

-   -   1. a backbone with a bacterial selectable marker (typically, an        antibiotic resistance gene) and origin of replication functional        in Escherichia coli (E. coli; e.g., ColE1), and    -   2. a plant-specific portion consisting of:        -   a. a gene expression cassette consisting of a promoter (eg.            ZmUBlint MOD), the gene of interest (typically, a            full-length cDNA) and a transcriptional terminator (e.g.,            Agrobacterium tumefaciens nos terminator);        -   b. a plant selectable marker cassette, consisting of a            suitable promoter, selectable marker gene (e.g., D-amino            acid oxidase; dao1) and transcriptional terminator (eg. nos            terminator).

Vectors designed for transformation by Agrobacterium tumefaciens (A.tumefaciens; binary vectors) consist of:

-   -   1. a backbone with a bacterial selectable marker functional in        both E. coli and A. tumefaciens (e.g., spectinomycin resistance        mediated by the aadA gene) and two origins of replication,        functional in each of aforementioned bacterial hosts, plus        the A. tumefaciens virG gene;    -   2. a plant-specific portion as described for biolistic vectors        above, except in this instance this portion is flanked by A.        tumefaciens right and left border sequences which mediate        transfer of the DNA flanked by these two sequences to the plant.

7.2 Gene Silencing Vectors

Vectors designed for reducing or abolishing expression of a single geneor of a family or related genes (gene silencing vectors) are also of twogeneral types corresponding to the methodology used to downregulate geneexpression: antisense or doublestranded RNA interference (dsRNAi).

(a) Anti-sense

For antisense vectors, a full-length or partial gene fragment(typically, a portion of the cDNA) can be used in the same vectorsdescribed for full-length expression, as part of the gene expressioncassette. For antisense-mediated down-regulation of gene expression, thecoding region of the gene or gene fragment will be in the oppositeorientation relative to the promoter; thus, mRNA will be made from thenon-coding (antisense) strand in planta.

(b) dsRNAi

For dsRNAi vectors, a partial gene fragment (typically, 300 to 500basepairs long) is used in the gene expression cassette, and isexpressed in both the sense and antisense orientations, separated by aspacer region (typically, a plant intron, eg. the OsSH1 intron 1, or aselectable marker, eg. conferring kanamycin resistance). Vectors of thistype are designed to form a double-stranded mRNA stem, resulting fromthe basepairing of the two complementary gene fragments in planta.

Biolistic or binary vectors designed for overexpression or knockout canvary in a number of different ways, including eg. the selectable markersused in plant and bacteria, the transcriptional terminators used in thegene expression and plant selectable marker cassettes, and themethodologies used for cloning in gene or gene fragments of interest(typically, conventional restriction enzyme-mediated or Gateway™recombinasebased cloning).

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1. An expression cassette for seed-specific or seed-preferentialtranscription in a plant comprising: i) at least one transcriptionregulating nucleotide sequence of a plant gene, and ii) at least onenucleic acid sequence which is operably linked to and heterologous inrelation to said transcription regulating nucleotide sequence; whereinthe plant gene comprises the nucleic acid sequence as set forth in SEQID NO: 48 and wherein the transcription regulating nucleotide sequencehas seed-specific or seed-preferential transcription activity.
 2. Anexpression cassette for seed-specific or seed-preferential transcriptionin a plant comprising: i) at least one transcription regulatingnucleotide sequence; and ii) at least one nucleic acid sequence which isoperably linked to and heterologous in relation to said transcriptionregulating nucleotide sequence; wherein the transcription regulatingnucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, or a nucleotide sequencehaving at least 99% identity to the nucleotide sequence of SEQ ID NO:36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, and wherein thetranscription regulating nucleotide sequence has seed-specific orseed-preferential transcription activity.
 3. The expression cassette ofclaim 1, wherein expression of the nucleic acid sequence results inexpression of a protein, or expression of an antisense RNA, a sense RNA,or a double-stranded RNA.
 4. The expression cassette of claim 1, whereinexpression of the at least one nucleic acid sequence confers to theplant an agronomically valuable trait.
 5. A vector comprising theexpression cassette of claim
 1. 6. A transgenic plant, plant cell ormicroorganism comprising the expression cassette of claim
 1. 7. Theexpression cassette of claim 2, wherein the transcription regulatingnucleotide sequence comprises a nucleotide sequence having at least 99%identity to the nucleotide sequence of SEQ ID NO: 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, or 47, and wherein the transcription regulatingnucleotide sequence has seed-specific or seed-preferential transcriptionactivity.
 8. The expression cassette of claim 2, wherein expression ofthe nucleic acid sequence results in expression of a protein, orexpression of an antisense RNA, a sense RNA, or a double-stranded RNA.9. The expression cassette of claim 2, wherein expression of the atleast one nucleic acid sequence confers to the plant an agronomicallyvaluable trait.
 10. A vector comprising the expression cassette of claim2.
 11. A transgenic plant, plant cell or microorganism comprising theexpression cassette of claim
 2. 12. An expression cassette forseed-specific or seed-preferential transcription in a plant comprising:i) at least one transcription regulating nucleotide sequence; and ii) atleast one nucleic acid sequence which is operably linked to andheterologous in relation to said transcription regulating nucleotidesequence; wherein the transcription regulating nucleotide sequencecomprises a fragment of SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, or 47, and wherein the fragment has seed-specific orseed-preferential expression activity.
 13. The expression cassette ofclaim 12, wherein expression of the nucleic acid sequence results inexpression of a protein, or expression of an antisense RNA, a sense RNA,or a double-stranded RNA.
 14. The expression cassette of claim 12,wherein expression of the at least one nucleic acid sequence confers tothe plant an agronomically valuable trait.
 15. A vector comprising theexpression cassette of claim
 12. 16. A transgenic plant, plant cell ormicroorganism comprising the expression cassette of claim
 12. 17. Amethod for producing a transgenic plant cell, which comprisestransforming a plant cell with the expression cassette of claim
 12. 18.A method for producing a transgenic plant, which comprises transforminga plant cell with the expression cassette of claim 12, and generatingfrom the plant cell the transgenic plant.